Team:CU-Boulder/Notebook/CC9 Team/Induced

From 2014.igem.org

(Difference between revisions)
Line 1: Line 1:
{{Template:UCB-Main}}
{{Template:UCB-Main}}
{{Template:UCB-NavBar}}
{{Template:UCB-NavBar}}
-
==Week 1==
+
Week 1
5/12/14   
5/12/14   
  Made overnight cultures of….
  Made overnight cultures of….
Line 12: Line 12:
Glycerol stocks  
Glycerol stocks  
- Stocks made from overnight cultures (3 from each)
- Stocks made from overnight cultures (3 from each)
-
- 500 μL bacteria + 500 μL 40% glycerol stock
+
- 500 ?L bacteria + 500 ?L 40% glycerol stock
*Glass tube containing #P44250 broke and was discarded – overnight repeated
*Glass tube containing #P44250 broke and was discarded – overnight repeated
Line 22: Line 22:
5/14/14
5/14/14
-
_t Nano Drop of Mini Prep
+
{| class = "wikitable"
-
Sample 260/280 Ratio 260/230 Ratio DNA Concentration
+
|-
-
P44250 1.94 2.17 49.4 ng/μL
+
!  Nano Drop of Mini Prep
-
P44251 1.94 2.47 52.7 ng/μL
+
!
-
_t
+
!
 +
!
 +
 
 +
|-
 +
| Sample
 +
| 260/280 Ratio
 +
| 260/230 Ratio
 +
| DNA Concentration
 +
 
 +
|-
 +
| P44250
 +
| 1.94
 +
| 2.17
 +
| 49.4 ng/?L
 +
 
 +
|-
 +
| P44251
 +
| 1.94
 +
| 2.47
 +
| 52.7 ng/?L
 +
 
 +
|-
 +
|}
*Samples were blanked with EB Buffer
*Samples were blanked with EB Buffer
Line 36: Line 58:
-
_t P44250 P44251
+
{| class = "wikitable"
-
Cutsmart Buffer 5.0 μL 5.0 μL
+
|-
-
XbaI 1.0 μL 1.0 μL
+
!
-
XhoI 1.0 μL --
+
! P44250
-
SpeI 1.0 μL --
+
! P44251
-
EcoRI-HF -- 1.0 μL
+
 
-
DNA (200 ng) 4.0 μL 4.0 μL
+
|-
-
MQ H2O 38.0 μL 39.0 μL
+
| Cutsmart Buffer
-
Total Volume 50.0 μL 50.0 μL
+
| 5.0 ?L
-
_t
+
| 5.0 ?L
 +
 
 +
|-
 +
| XbaI
 +
| 1.0 ?L
 +
| 1.0 ?L
 +
 
 +
|-
 +
| XhoI
 +
| 1.0 ?L
 +
| --
 +
 
 +
|-
 +
| SpeI
 +
| 1.0 ?L
 +
| --
 +
 
 +
|-
 +
| EcoRI-HF
 +
| --
 +
| 1.0 ?L
 +
 
 +
|-
 +
| DNA (200 ng)
 +
| 4.0 ?L
 +
| 4.0 ?L
 +
 
 +
|-
 +
| MQ H2O
 +
| 38.0 ?L
 +
| 39.0 ?L
 +
 
 +
|-
 +
| Total Volume
 +
| 50.0 ?L
 +
| 50.0 ?L
 +
 
 +
|-
 +
|}
Agarose Gel Electrophoresis
Agarose Gel Electrophoresis
-1% agarose gel prepared  
-1% agarose gel prepared  
-
-2 μL loading dye added to 18 μL of each sample for loading into each lane
+
-2 ?L loading dye added to 18 ?L of each sample for loading into each lane
-1Kb+ DNA Ladder used
-1Kb+ DNA Ladder used
-Overnight staining in EtBr done to better visualize faint bands
-Overnight staining in EtBr done to better visualize faint bands
Line 65: Line 125:
Plate Streaking
Plate Streaking
-Used to check the TetR gene in the P44250 plasmid
-Used to check the TetR gene in the P44250 plasmid
-
-2 mL LB containing 50 mg/ μL Tet added to plate and allowed to soak in
+
-2 mL LB containing 50 mg/ ?L Tet added to plate and allowed to soak in
-Plates left at room temperature overnight
-Plates left at room temperature overnight
Line 97: Line 157:
5/19/14
5/19/14
Transformation:
Transformation:
-
1 μL P44250 (cas9) plasmid added to K12 E. coli strain
+
1 ?L P44250 (cas9) plasmid added to K12 E. coli strain
Heat shock done according to protocol (45 sec @ 42°C)
Heat shock done according to protocol (45 sec @ 42°C)
Cultures incubated at 37°C for 1.5 hours
Cultures incubated at 37°C for 1.5 hours
4 Plates with K12 and the plasmid were plated by streaking as follows:
4 Plates with K12 and the plasmid were plated by streaking as follows:
-
- Amp, Amp (½ plate with 1:10 dilution, ½ plate 1:1), Amp + Kan, Amp + Kan (½ plate with 1:10 dilution, ½ plate 1:1)
+
- Amp, Amp (? plate with 1:10 dilution, ? plate 1:1), Amp + Kan, Amp + Kan (? plate with 1:10 dilution, ? plate 1:1)
5/20/14
5/20/14
Plates from 5/19/14 (shown below).
Plates from 5/19/14 (shown below).
Line 112: Line 172:
5/22/14
5/22/14
Transformation: (Chemically competent BWF+ cells with P44250 plasmid)
Transformation: (Chemically competent BWF+ cells with P44250 plasmid)
-
1 μL P44250 (cas9) plasmid added to conjugated BWF+ strain
+
1 ?L P44250 (cas9) plasmid added to conjugated BWF+ strain
Transformation protocol followed with following exceptions:
Transformation protocol followed with following exceptions:
-
-200 μL SOC added to each sample  
+
-200 ?L SOC added to each sample  
-Incubated for 2 hours
-Incubated for 2 hours
-
-1:10 dilution made (10 μL into 90 μL SOC)
+
-1:10 dilution made (10 ?L into 90 ?L SOC)
-Cultures plated on Amp plates, one with full concentration and another with the 1:10 dilution
-Cultures plated on Amp plates, one with full concentration and another with the 1:10 dilution
Line 122: Line 182:
5/23/14
5/23/14
Arrival of primers previously ordered
Arrival of primers previously ordered
-
All were reconstituted in MQ H2O to 100 μM concentration
+
All were reconstituted in MQ H2O to 100 ?M concentration
Results of plates from 5/22/14:
Results of plates from 5/22/14:
Line 133: Line 193:
-
Single colonies taken from the BFW+ w/ P50 plasmid plate in the 1:10 dilution and placed in 2 μL liquid culture overnight with the following antibiotics:
+
Single colonies taken from the BFW+ w/ P50 plasmid plate in the 1:10 dilution and placed in 2 ?L liquid culture overnight with the following antibiotics:
- AMP
- AMP
- Kan
- Kan
Line 146: Line 206:
PCR Reaction – Used to amplify out cas9 from P44250 plasmid and CMR from PSB1C3 plasmid
PCR Reaction – Used to amplify out cas9 from P44250 plasmid and CMR from PSB1C3 plasmid
-
*DNA samples diluted so each contains 0.5 ng/μL
+
*DNA samples diluted so each contains 0.5 ng/?L
-
_t Cas9 CMR
+
{| class = "wikitable"
-
Q5 Master Mix volume 25 μL 25 μL
+
|-
-
dcas9 Primer volume 2.5 μL --
+
!
-
dCMR Primer volume -- 2.5 μL
+
! Cas9
-
DNA added 2.5 μL 2.5 μL
+
! CMR
-
Q5 Polymerase volume 0.5 μL 0.5 μL
+
 
-
Nuclease Free H2O 20 μL 20 μL
+
|-
-
Final Reaction Volume 50.0 μL 50.0 μL
+
| Q5 Master Mix volume
-
_t
+
| 25 ?L
 +
| 25 ?L
 +
 
 +
|-
 +
| dcas9 Primer volume
 +
| 2.5 ?L
 +
| --
 +
 
 +
|-
 +
| dCMR Primer volume
 +
| --
 +
| 2.5 ?L
 +
 
 +
|-
 +
| DNA added
 +
| 2.5 ?L
 +
| 2.5 ?L
 +
 
 +
|-
 +
| Q5 Polymerase volume
 +
| 0.5 ?L
 +
| 0.5 ?L
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 20 ?L
 +
| 20 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 50.0 ?L
 +
| 50.0 ?L
 +
 
 +
|-
 +
|}
PCR Settings: 30 cycles as follows
PCR Settings: 30 cycles as follows
- 98 °C Denature 30 sec
- 98 °C Denature 30 sec
Line 165: Line 259:
5/25/14
5/25/14
-
Gel electrophoresis performed on PCR reactions from 5/24/14. 1% gel made from 0.514 g agarose with 50 mL 1X TAE and 5 μL EtBr.  
+
Gel electrophoresis performed on PCR reactions from 5/24/14. 1% gel made from 0.514 g agarose with 50 mL 1X TAE and 5 ?L EtBr.  
-
Lane 1: 5 μL 2-Log DNA Ladder
+
Lane 1: 5 ?L 2-Log DNA Ladder
Lane 2: CMR sample* (~2 Kb size)
Lane 2: CMR sample* (~2 Kb size)
Lane 3: Cas9 sample* (~5 Kb size)
Lane 3: Cas9 sample* (~5 Kb size)
-
*Each contained 8 μL reaction sample + 2 μL loading dye  
+
*Each contained 8 ?L reaction sample + 2 ?L loading dye  
Gel Results:  
Gel Results:  
Line 183: Line 277:
5/27/14
5/27/14
Repeat of PCR using same protocol as listed on 5/24/14 except for different times on the thermocycler as follows:
Repeat of PCR using same protocol as listed on 5/24/14 except for different times on the thermocycler as follows:
-
_t Cas9 CMR
+
{| class = "wikitable"
-
Initial Denature 98°C/2 min 98°C/2 min
+
|-
-
Denature 98°C/30 sec 98°C/30 sec
+
!
-
Anneal 69°C/30 sec 72°C/30 sec
+
! Cas9
-
Extend 69°C/ 2.5 min 72°C/2.5 min
+
! CMR
-
Final Extension 72°C/6 min 72°C/5 min
+
 
-
_t
+
|-
 +
| Initial Denature
 +
| 98°C/2 min
 +
| 98°C/2 min
 +
 
 +
|-
 +
| Denature
 +
| 98°C/30 sec
 +
| 98°C/30 sec
 +
 
 +
|-
 +
| Anneal
 +
| 69°C/30 sec
 +
| 72°C/30 sec
 +
 
 +
|-
 +
| Extend
 +
| 69°C/ 2.5 min
 +
| 72°C/2.5 min
 +
 
 +
|-
 +
| Final Extension
 +
| 72°C/6 min
 +
| 72°C/5 min
 +
 
 +
|-
 +
|}
* Thermocycler containing CMR stopped after 1 cycle and held at 98°C for ~3hours. Machine was reset and run. This sample may no longer be viable.
* Thermocycler containing CMR stopped after 1 cycle and held at 98°C for ~3hours. Machine was reset and run. This sample may no longer be viable.
Line 196: Line 316:
Dry lab – primer designs
Dry lab – primer designs
Repeat of PCR for the CMR reaction, same as described on 5/24/14 and shown below.
Repeat of PCR for the CMR reaction, same as described on 5/24/14 and shown below.
-
_t Reactants CMR
+
{| class = "wikitable"
-
Q5 Master Mix 25 μL
+
|-
-
dCMR Primer 2.5 μL
+
!  Reactants
-
Q5 Polymerase 0.5 μL
+
! CMR
-
CMR DNA 2.0 μL
+
 
-
Nuclease Free H2O 20 μL
+
|-
-
Final Reaction Volume 50.0 μL
+
| Q5 Master Mix  
-
_t
+
| 25 ?L
 +
 
 +
|-
 +
| dCMR Primer  
 +
| 2.5 ?L
 +
 
 +
|-
 +
| Q5 Polymerase  
 +
| 0.5 ?L
 +
 
 +
|-
 +
| CMR DNA
 +
| 2.0 ?L
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 20 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 50.0 ?L
 +
 
 +
|-
 +
|}
5/29/14
5/29/14
Gel electrophoresis: 1% gel ran at 110V for ~40 minutes.
Gel electrophoresis: 1% gel ran at 110V for ~40 minutes.
-
_t Lane Sample Amount Loading dye amount
+
{| class = "wikitable"
-
1 2-Log DNA Ladder 0.5  μL --
+
|-
-
2 Cas9 5.0  μL 1.0  μL
+
!  Lane
-
3 CMR 1 (from 5/24) 5.0  μL 1.0  μL
+
! Sample
-
4 CMR 2 (from 5/27) 5.0  μL 1.0  μL
+
! Amount
-
5 CMR 3 (from 5/28) 5.0  μL 1.0  μL
+
! Loading dye amount
-
_t
+
 
 +
|-
 +
| 1
 +
| 2-Log DNA Ladder
 +
| 0.5  ?L
 +
| --
 +
 
 +
|-
 +
| 2
 +
| Cas9
 +
| 5.0  ?L
 +
| 1.0  ?L
 +
 
 +
|-
 +
| 3
 +
| CMR 1 (from 5/24)
 +
| 5.0  ?L
 +
| 1.0  ?L
 +
 
 +
|-
 +
| 4
 +
| CMR 2 (from 5/27)
 +
| 5.0  ?L
 +
| 1.0  ?L
 +
 
 +
|-
 +
| 5
 +
| CMR 3 (from 5/28)
 +
| 5.0  ?L
 +
| 1.0  ?L
 +
 
 +
|-
 +
|}
Results from Gel (shown below):  
Results from Gel (shown below):  
Lane 2: Cas9 band visible of appropriate size (approximately 5,000 bp)
Lane 2: Cas9 band visible of appropriate size (approximately 5,000 bp)
Line 224: Line 399:
Gel extraction performed on cas9, CMR1, and CMR2 lanes using E.Z.N.A. Gel extraction kit following  the protocol. Following amounts:
Gel extraction performed on cas9, CMR1, and CMR2 lanes using E.Z.N.A. Gel extraction kit following  the protocol. Following amounts:
-
- Cas9: 110 μL buffer with 0.1103 g sample
+
- Cas9: 110 ?L buffer with 0.1103 g sample
-
- CMR1: 53 μL buffer with 0.0533 g sample
+
- CMR1: 53 ?L buffer with 0.0533 g sample
-
- CMR2: 90 μL buffer with 0.0904 g sample
+
- CMR2: 90 ?L buffer with 0.0904 g sample
Nanodrops performed to determine DNA concentration and purity with the following results:
Nanodrops performed to determine DNA concentration and purity with the following results:
-
_t Sample ng/ μL 260/280 260/230 **ng/ μL **260/280 **260/230
+
{| class = "wikitable"
-
Gel-Cas9 7.3 2.20 0.57 5.2 2.06 0.52
+
|-
-
Cas9 483.2 1.78 0.72 36.3 1.85 1.72
+
!  Sample
-
Gel-CMR1 9.6 2.02 0.63 6.5 2.35 0.90
+
! ng/ ?L
-
Gel-CMR2 8.3 2.06 0.83 5.0 2.21 0.69
+
! 260/280
-
CMR2 618.8 1.66 0.61 44.5 1.89 1.41
+
! 260/230
-
CMR 3 535.8 1.73 0.61 17.8 1.89 1.31
+
! **ng/ ?L
-
CMR1 516.6 1.69 0.61 68.5 1.84 1.74
+
! **260/280
-
_t
+
! **260/230
 +
 
 +
|-
 +
| Gel-Cas9
 +
| 7.3
 +
| 2.20
 +
| 0.57
 +
| 5.2
 +
| 2.06
 +
| 0.52
 +
 
 +
|-
 +
| Cas9
 +
| 483.2
 +
| 1.78
 +
| 0.72
 +
| 36.3
 +
| 1.85
 +
| 1.72
 +
 
 +
|-
 +
| Gel-CMR1
 +
| 9.6
 +
| 2.02
 +
| 0.63
 +
| 6.5
 +
| 2.35
 +
| 0.90
 +
 
 +
|-
 +
| Gel-CMR2
 +
| 8.3
 +
| 2.06
 +
| 0.83
 +
| 5.0
 +
| 2.21
 +
| 0.69
 +
 
 +
|-
 +
| CMR2
 +
| 618.8
 +
| 1.66
 +
| 0.61
 +
| 44.5
 +
| 1.89
 +
| 1.41
 +
 
 +
|-
 +
| CMR 3
 +
| 535.8
 +
| 1.73
 +
| 0.61
 +
| 17.8
 +
| 1.89
 +
| 1.31
 +
 
 +
|-
 +
| CMR1
 +
| 516.6
 +
| 1.69
 +
| 0.61
 +
| 68.5
 +
| 1.84
 +
| 1.74
 +
 
 +
|-
 +
|}
*Trial 2 completed on 5/30/14 shown in these columns
*Trial 2 completed on 5/30/14 shown in these columns
Values for the ratios should be between 2.0 and 2.2 for a pure sample. The values for Trial #1 are much too low. In order to increase sample purity, a DNA cleanup is performed following the protocol given in the E.Z.N.A. gel extraction kit in amounts shown below.
Values for the ratios should be between 2.0 and 2.2 for a pure sample. The values for Trial #1 are much too low. In order to increase sample purity, a DNA cleanup is performed following the protocol given in the E.Z.N.A. gel extraction kit in amounts shown below.
-
- 25 μL samples
+
- 25 ?L samples
-
- 25 μL binding buffer
+
- 25 ?L binding buffer
-
- Washed 2x with 700 μL SPW
+
- Washed 2x with 700 ?L SPW
-
- 30 μL elution buffer added, allowed to rest 4 minutes
+
- 30 ?L elution buffer added, allowed to rest 4 minutes
- Centrifuged on max for 2 min
- Centrifuged on max for 2 min
Line 254: Line 495:
Restriction Digestion performed on Cas9 and CMR1 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts:
Restriction Digestion performed on Cas9 and CMR1 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts:
-
26 μL sample
+
26 ?L sample
-
1.0 μL DPN1 (100 fold dilution)
+
1.0 ?L DPN1 (100 fold dilution)
-
3.0 μL 10X CutSmart Buffer  
+
3.0 ?L 10X CutSmart Buffer  
Gibson Assembly performed to join pSB1C3 backbone with cas9 PCR product. Protocol for Gibson was followed for the reaction.
Gibson Assembly performed to join pSB1C3 backbone with cas9 PCR product. Protocol for Gibson was followed for the reaction.
-
_t Reactants Volume
+
{| class = "wikitable"
-
Gibson Master Mix 10 μL
+
|-
-
Cas9 1.6 μL (50 ng)
+
!  Reactants
-
Cmr 2.4 μL (143 ng)
+
! Volume
-
Nuclease Free H2O 6 μL
+
 
-
Final Reaction Volume 20.0 μL
+
|-
-
_t
+
| Gibson Master Mix  
 +
| 10 ?L
 +
 
 +
|-
 +
| Cas9
 +
| 1.6 ?L (50 ng)
 +
 
 +
|-
 +
| Cmr
 +
| 2.4 ?L (143 ng)
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 6 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 20.0 ?L
 +
 
 +
|-
 +
|}
Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.  
Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.  
Line 273: Line 534:
6/2/14
6/2/14
-
Grow Gibson colonies in 6 mL LB media + 30 μL chloroamp
+
Grow Gibson colonies in 6 mL LB media + 30 ?L chloroamp
Transform Gibson colonies following protocol and with amounts as shown:
Transform Gibson colonies following protocol and with amounts as shown:
-
- BWF+ w/cas9: 15 μL into 135 μL SOC
+
- BWF+ w/cas9: 15 ?L into 135 ?L SOC
-
- BW w/cas9: 15 μL into 135 μL SOC
+
- BW w/cas9: 15 ?L into 135 ?L SOC
-
- BWF+ (no cas9):  30 μL into 270 μL SOC
+
- BWF+ (no cas9):  30 ?L into 270 ?L SOC
-
- BW (no cas9): 30 μL into 270 μL SOC
+
- BW (no cas9): 30 ?L into 270 ?L SOC
-
90 μL of the above four reactions plated as follows and incubated:
+
90 ?L of the above four reactions plated as follows and incubated:
- BWF+ w/cas9: Amp + Kan
- BWF+ w/cas9: Amp + Kan
- BW w/cas9: Amp + Kan  
- BW w/cas9: Amp + Kan  
Line 297: Line 558:
NanoPrep on GAcasC
NanoPrep on GAcasC
-
_t ng/ μL 260/280 260/230
+
{| class = "wikitable"
-
201.0 1.88 2.24
+
|-
-
_t
+
!  ng/ ?L
 +
! 260/280
 +
! 260/230
 +
 
 +
|-
 +
| 201.0
 +
| 1.88
 +
| 2.24
 +
 
 +
|-
 +
|}
Digest performed according to protocol on GAcasC and P50 plasmid using EcoRV and BamHI as follows:  
Digest performed according to protocol on GAcasC and P50 plasmid using EcoRV and BamHI as follows:  
-
_t Reactants GAcasC P50
+
{| class = "wikitable"
-
Buffer 3 2.5 μL 2.5 μL
+
|-
-
EcoRV 0.5 μL 0.5 μL
+
!  Reactants
-
BamHI 0.5 μL 0.5 μL
+
! GAcasC
-
BSA (1:4 dilution) 1.0 μL 1.0 μL
+
! P50
-
DNA (250 ng) 1.25 μL 5.0 μL
+
 
-
MQ H2O 19.25 μL 15.5 μL
+
|-
-
Total Volume 25.0 μL 25.0 μL
+
| Buffer 3
-
_t
+
| 2.5 ?L
 +
| 2.5 ?L
 +
 
 +
|-
 +
| EcoRV
 +
| 0.5 ?L
 +
| 0.5 ?L
 +
 
 +
|-
 +
| BamHI
 +
| 0.5 ?L
 +
| 0.5 ?L
 +
 
 +
|-
 +
| BSA (1:4 dilution)  
 +
| 1.0 ?L
 +
| 1.0 ?L
 +
 
 +
|-
 +
| DNA (250 ng)
 +
| 1.25 ?L
 +
| 5.0 ?L
 +
 
 +
|-
 +
| MQ H2O
 +
| 19.25 ?L
 +
| 15.5 ?L
 +
 
 +
|-
 +
| Total Volume
 +
| 25.0 ?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
|}
 +
 
 +
1% Gel made for electrophoresis. Each sample had 1 ?L of loading dye added, 5 ?L of DNA ladder and 5 ?L of each sample was loaded. Gel ran 120V for ~1 hour.
 +
 
 +
{| class = "wikitable"
 +
|-
 +
!  Well
 +
! Loaded
 +
 
 +
|-
 +
| 1
 +
| DNA Ladder
 +
 
 +
|-
 +
| 2
 +
| GAcasC digest
 +
 
 +
|-
 +
| 3
 +
| CmR1 Dpn1 product
 +
 
 +
|-
 +
| 4
 +
| Cas9 Dpn1 product
-
1% Gel made for electrophoresis. Each sample had 1 μL of loading dye added, 5 μL of DNA ladder and 5 μL of each sample was loaded. Gel ran 120V for ~1 hour.
+
|-
 +
| 5
 +
| P50 digestion product
-
_t Well Loaded
+
|-
-
1 DNA Ladder
+
|}
-
2 GAcasC digest
+
-
3 CmR1 Dpn1 product
+
-
4 Cas9 Dpn1 product
+
-
5 P50 digestion product
+
-
_t
+
Gel Results:
Gel Results:
Lane 2 should have multiple products which are not present in the gel. The other lanes are as expected.  
Lane 2 should have multiple products which are not present in the gel. The other lanes are as expected.  
Line 329: Line 654:
PCR using Q5 is used to amplify the P51 CRISPR backbone using primers CRE1 & CRE4 provided by Andrew. Primers were diluted 1:10 from 100 mM stock
PCR using Q5 is used to amplify the P51 CRISPR backbone using primers CRE1 & CRE4 provided by Andrew. Primers were diluted 1:10 from 100 mM stock
-
_t Reactants Volume
+
{| class = "wikitable"
-
Q5 Master Mix 25 μL
+
|-
-
CRE1 Primer 2.5 μL
+
!  Reactants
-
CRE4 Primer 2.5 μL
+
! Volume
-
P51 DNA 2.0 μL
+
 
-
Q5 Polymerase 0.5 μL
+
|-
-
Nuclease Free H2O 17.5 μL
+
| Q5 Master Mix  
-
Final Reaction Volume 50.0 μL
+
| 25 ?L
-
_t
+
 
 +
|-
 +
| CRE1 Primer
 +
| 2.5 ?L
 +
 
 +
|-
 +
| CRE4 Primer
 +
| 2.5 ?L
 +
 
 +
|-
 +
| P51 DNA
 +
| 2.0 ?L
 +
 
 +
|-
 +
| Q5 Polymerase  
 +
| 0.5 ?L
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 17.5 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 50.0 ?L
 +
 
 +
|-
 +
|}
Thermocycler set as follows for the reaction:
Thermocycler set as follows for the reaction:
-
_t Initial Denature 98°C/30 sec
+
{| class = "wikitable"
-
Denature 98°C/20 sec
+
|-
-
Anneal 60°C/20 sec
+
!  Initial Denature
-
Extend 72°C/3 min
+
! 98°C/30 sec
-
_t
+
 
 +
|-
 +
| Denature
 +
| 98°C/20 sec
 +
 
 +
|-
 +
| Anneal
 +
| 60°C/20 sec
 +
 
 +
|-
 +
| Extend
 +
| 72°C/3 min
 +
 
 +
|-
 +
|}
Restriction Digestion performed on P51 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts:
Restriction Digestion performed on P51 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts:
-
26 μL sample
+
26 ?L sample
-
1.0 μL DPN1 (100 fold dilution)
+
1.0 ?L DPN1 (100 fold dilution)
-
3.0 μL 10X CutSmart Buffer  
+
3.0 ?L 10X CutSmart Buffer  
PCR performed to dimerize each of the gRNA primers previously designed (729, 666, 756, SC1, and SC2) following the cPEC protocol and using the following amounts:
PCR performed to dimerize each of the gRNA primers previously designed (729, 666, 756, SC1, and SC2) following the cPEC protocol and using the following amounts:
-
_t Reactants Volume
+
{| class = "wikitable"
-
Q5 Master Mix 25.0 μL
+
|-
-
Primer 1.7 μL
+
!  Reactants
-
Primer 2.0 μL
+
! Volume
-
Q5 Polymerase 0. 5 μL
+
 
-
Nuclease Free H2O 19.5 μL
+
|-
-
Final Reaction Volume 50.0 μL
+
| Q5 Master Mix  
-
_t
+
| 25.0 ?L
 +
 
 +
|-
 +
| Primer
 +
| 1.7 ?L
 +
 
 +
|-
 +
| Primer  
 +
| 2.0 ?L
 +
 
 +
|-
 +
| Q5 Polymerase  
 +
| 0. 5 ?L
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 19.5 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 50.0 ?L
 +
 
 +
|-
 +
|}
Nanodrop performed on some of these products (due to timing) to check for purity and concentration (results below). It is assumed that the other samples have high enough DNA levels to proceed.
Nanodrop performed on some of these products (due to timing) to check for purity and concentration (results below). It is assumed that the other samples have high enough DNA levels to proceed.
-
_t Sample ng/ μL 260/280 260/230
+
{| class = "wikitable"
-
729 405.5 1.78 0.67
+
|-
-
SC2 393.2 1.80 0.67
+
!  Sample
-
CasC 455.2 1.5 0.60
+
! ng/ ?L
-
_t
+
! 260/280
 +
! 260/230
 +
 
 +
|-
 +
| 729
 +
| 405.5
 +
| 1.78
 +
| 0.67
 +
 
 +
|-
 +
| SC2
 +
| 393.2
 +
| 1.80
 +
| 0.67
 +
 
 +
|-
 +
| CasC
 +
| 455.2
 +
| 1.5
 +
| 0.60
 +
 
 +
|-
 +
|}
cPEC performed to combine the P51 amplification and the each of the dimerized gRNAs separately following the cPEC protocol and using the following amounts:
cPEC performed to combine the P51 amplification and the each of the dimerized gRNAs separately following the cPEC protocol and using the following amounts:
Line 375: Line 785:
-
_t Reactants Volume
+
{| class = "wikitable"
-
Q5 Master Mix 12.5 μL
+
|-
-
Insert (our primer dimers) 1.7 μL
+
!  Reactants
-
Template DNA 2.0 μL
+
! Volume
-
Q5 Polymerase 0.25 μL
+
 
-
Nuclease Free H2O 10.05 μL
+
|-
-
Final Reaction Volume 25.0 μL
+
| Q5 Master Mix  
-
_t
+
| 12.5 ?L
 +
 
 +
|-
 +
| Insert (our primer dimers)
 +
| 1.7 ?L
 +
 
 +
|-
 +
| Template DNA
 +
| 2.0 ?L
 +
 
 +
|-
 +
| Q5 Polymerase  
 +
| 0.25 ?L
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 10.05 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 25.0 ?L
 +
 
 +
|-
 +
|}
Repeat of Gibson to combine cas9 and CmR (pSB1C3 backbone) with cas9 PCR product. Gibson protocol was followed for the reaction and included a positive control (same as reaction completed on 5/30/14).
Repeat of Gibson to combine cas9 and CmR (pSB1C3 backbone) with cas9 PCR product. Gibson protocol was followed for the reaction and included a positive control (same as reaction completed on 5/30/14).
-
_t Reactants Volume
+
{| class = "wikitable"
-
Gibson Master Mix 10 μL
+
|-
-
Cas9 1.6 μL (50 ng)
+
!  Reactants
-
Cmr 2.4 μL (143 ng)
+
! Volume
-
Nuclease Free H2O 6 μL
+
 
-
Final Reaction Volume 20.0 μL
+
|-
-
_t
+
| Gibson Master Mix  
 +
| 10 ?L
 +
 
 +
|-
 +
| Cas9
 +
| 1.6 ?L (50 ng)
 +
 
 +
|-
 +
| Cmr
 +
| 2.4 ?L (143 ng)
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 6 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 20.0 ?L
 +
 
 +
|-
 +
|}
Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.  
Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.  
Line 398: Line 851:
O/N cell cultures made from plates in order to perform a Tet titration tomorrow. 2 tubes of each were made by taking a single colony and mixing with 5 mL of LB and antibiotics as listed below before incubation.  
O/N cell cultures made from plates in order to perform a Tet titration tomorrow. 2 tubes of each were made by taking a single colony and mixing with 5 mL of LB and antibiotics as listed below before incubation.  
-
_t Sample Antibiotic
+
{| class = "wikitable"
-
BWF+ w/ cas 5 μL Kan + 10 μL Amp
+
|-
-
BW w/ cas 5 μL Kan + 10 μL Amp
+
!  Sample
-
BWF+ 5 μL Kan
+
! Antibiotic
-
BW 5 μL Kan
+
 
-
BWF+ (not transformed) 5 μL Kan + 10 μL Tet
+
|-
-
_t
+
| BWF+ w/ cas
 +
| 5 ?L Kan + 10 ?L Amp
 +
 
 +
|-
 +
| BW w/ cas
 +
| 5 ?L Kan + 10 ?L Amp
 +
 
 +
|-
 +
| BWF+
 +
| 5 ?L Kan
 +
 
 +
|-
 +
| BW
 +
| 5 ?L Kan
 +
 
 +
|-
 +
| BWF+ (not transformed)
 +
| 5 ?L Kan + 10 ?L Tet
 +
 
 +
|-
 +
|}
6/4/14
6/4/14
Line 411: Line 884:
*Cancelled as cell growth had already neared plateau values  
*Cancelled as cell growth had already neared plateau values  
-
_t Sample 0 ng/mL 0.5 ng/mL 5 ng/mL 20 ng/mL 50 ng/mL 250 ng/mL 1000 ng/mL Chloro
+
{| class = "wikitable"
-
BW 2.38 2.401 2.389 2.382 2.386 2.398 2.373 2.379
+
|-
-
BWF+ 2.379 2.376 2.428 2.390 2.378 2.365 2.378 2.386
+
!  Sample
-
BWcas 2.391 2.356 2.367 2.355 2.366 2.330 2.362 2.382
+
! 0 ng/mL
-
BWF+ cas 2.438 2.447 2.448 2.434 2.436 2.415 2.419 2.415
+
! 0.5 ng/mL
-
BWF+ Tet 2.135 2.146 2.141 2.152 2.128 2.127 2.134 2.134
+
! 5 ng/mL
-
_t
+
! 20 ng/mL
 +
! 50 ng/mL
 +
! 250 ng/mL
 +
! 1000 ng/mL
 +
! Chloro
 +
 
 +
|-
 +
| BW
 +
| 2.38
 +
| 2.401
 +
| 2.389
 +
| 2.382
 +
| 2.386
 +
| 2.398
 +
| 2.373
 +
| 2.379
 +
 
 +
|-
 +
| BWF+
 +
| 2.379
 +
| 2.376
 +
| 2.428
 +
| 2.390
 +
| 2.378
 +
| 2.365
 +
| 2.378
 +
| 2.386
 +
 
 +
|-
 +
| BWcas
 +
| 2.391
 +
| 2.356
 +
| 2.367
 +
| 2.355
 +
| 2.366
 +
| 2.330
 +
| 2.362
 +
| 2.382
 +
 
 +
|-
 +
| BWF+ cas
 +
| 2.438
 +
| 2.447
 +
| 2.448
 +
| 2.434
 +
| 2.436
 +
| 2.415
 +
| 2.419
 +
| 2.415
 +
 
 +
|-
 +
| BWF+ Tet
 +
| 2.135
 +
| 2.146
 +
| 2.141
 +
| 2.152
 +
| 2.128
 +
| 2.127
 +
| 2.134
 +
| 2.134
 +
 
 +
|-
 +
|}
New O/N cell cultures made to do the titration on 6/5/14. Same amounts as shown above were used except only one of each was made.  No cells added to O/N culture on 6/4/14, that is why procedure was repeated.
New O/N cell cultures made to do the titration on 6/5/14. Same amounts as shown above were used except only one of each was made.  No cells added to O/N culture on 6/4/14, that is why procedure was repeated.
Line 426: Line 961:
   
   
   
   
-
_t Reactants Volume
+
{| class = "wikitable"
-
Q5 Master Mix 10.0 μL
+
|-
-
Cas9 DNA 5.0 μL
+
!  Reactants
-
CmR DNA 5.0 μL
+
! Volume
-
Nuclease Free H2O 0.00 μL
+
-
Final Reaction Volume 20.0 μL
+
-
_t
+
|-
 +
| Q5 Master Mix
 +
| 10.0 ?L
 +
 
 +
|-
 +
| Cas9 DNA
 +
| 5.0 ?L
 +
 
 +
|-
 +
| CmR DNA
 +
| 5.0 ?L
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 0.00 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 20.0 ?L
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
Thermocycler was varied for each of the three cPEC reactions as listed below:
Thermocycler was varied for each of the three cPEC reactions as listed below:
   
   
-
_t cPEC1 cPEC2 cPEC3
+
{| class = "wikitable"
-
Initial Denature 98°C/30 sec 98°C/30 sec 98°C/30 sec
+
|-
-
Denature 98°C/10 sec 98°C/20 sec 98°C/10 sec
+
!
-
Anneal 56°C/30 sec 60°C/20 sec 66°C/30 sec
+
! cPEC1
-
Extend 72°C/90 sec 72°C/30 sec 72°C/90 sec
+
! cPEC2
-
# of cycles 10 15 10
+
! cPEC3
-
Final Extension 72°C/10 min 72°C/10 min 72°C/10 min
+
 
-
  _t
+
|-
-
   
+
| Initial Denature
-
Nanodrop done on the three cPEC casC reactions with results listed below:
+
| 98°C/30 sec
 +
| 98°C/30 sec
 +
| 98°C/30 sec
 +
 
 +
|-
 +
| Denature
 +
| 98°C/10 sec
 +
| 98°C/20 sec
 +
| 98°C/10 sec
 +
 
 +
|-
 +
| Anneal
 +
| 56°C/30 sec
 +
| 60°C/20 sec
 +
| 66°C/30 sec
 +
 
 +
|-
 +
| Extend
 +
| 72°C/90 sec
 +
| 72°C/30 sec
 +
| 72°C/90 sec
 +
 
 +
|-
 +
| # of cycles
 +
| 10
 +
| 15
 +
| 10
 +
 
 +
|-
 +
| Final Extension
 +
| 72°C/10 min
 +
| 72°C/10 min
 +
| 72°C/10 min
 +
 
 +
|-
 +
| _t
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
| Nanodrop done on the three cPEC casC reactions with results listed below:
 +
 
 +
|-
 +
|
-
_t Sample ng/ μL 260/280 260/230
+
|-
 +
|}
CasC1 489.3 1.80 0.72
CasC1 489.3 1.80 0.72
CasC2 577.7 1.76 0.59
CasC2 577.7 1.76 0.59
Line 456: Line 1,058:
-
_t Sample ng/ μL 260/280 260/230
+
{| class = "wikitable"
-
CasC1 13.4 1.99 1.61
+
|-
-
CasC2 11.1 2.02 1.15
+
!  Sample
-
CasC3* 9.9 1.84 1.21
+
! ng/ ?L
-
  _t  
+
! 260/280
-
1% Gel made for electrophoresis. Each sample had 1 μL of loading dye added, 5 μL of DNA ladder and 5 μL of each sample was loaded. Gel ran 122V for ~50 min.
+
! 260/230
-
   
+
 
-
_t Well Loaded
+
|-
 +
| CasC1
 +
| 13.4
 +
| 1.99
 +
| 1.61
 +
 
 +
|-
 +
| CasC2
 +
| 11.1
 +
| 2.02
 +
| 1.15
 +
 
 +
|-
 +
| CasC3*
 +
| 9.9
 +
| 1.84
 +
| 1.21
 +
 
 +
|-
 +
| _t  
 +
 
 +
|-
 +
| 1% Gel made for electrophoresis. Each sample had 1 ?L of loading dye added, 5 ?L of DNA ladder and 5 ?L of each sample was loaded. Gel ran 122V for ~50 min.
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
|}
1 2-Log DNA Ladder
1 2-Log DNA Ladder
2 cPEC1
2 cPEC1
Line 478: Line 1,108:
Titration experiment was done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. It was decided to use BWF+ w/cas9 and w/o cas9 for this experiment. Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.
Titration experiment was done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. It was decided to use BWF+ w/cas9 and w/o cas9 for this experiment. Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.
   
   
-
_t Time 0 Time 1 Time 2 Time 3 Time 4 Time 5* Time 6
+
{| class = "wikitable"
-
BWF+ 0.041 0.047 0.114 0.283 0.597 0.065 0.171
+
|-
-
BWF+ w/cas9 0.035 0.046 0.110 0.262 0.530 0.059 0.154
+
! 
-
_t
+
! Time 0
 +
! Time 1
 +
! Time 2
 +
! Time 3
 +
! Time 4
 +
! Time 5*
 +
! Time 6
 +
 
 +
|-
 +
| BWF+
 +
| 0.041
 +
| 0.047
 +
| 0.114
 +
| 0.283
 +
| 0.597
 +
| 0.065
 +
| 0.171
 +
 
 +
|-
 +
| BWF+ w/cas9
 +
| 0.035
 +
| 0.046
 +
| 0.110
 +
| 0.262
 +
| 0.530
 +
| 0.059
 +
| 0.154
 +
 
 +
|-
 +
|}
*At Time 5, the cultures were again diluted down. The experiment was cancelled after Time 6 due to time constraints and issues with the plates.
*At Time 5, the cultures were again diluted down. The experiment was cancelled after Time 6 due to time constraints and issues with the plates.
Line 500: Line 1,159:
   
   
-
_t Sample ng/ μL 260/280 260/230
+
{| class = "wikitable"
-
GA1 80.5 1.88 2.04
+
|-
-
GA2 87.8 1.90 2.14
+
!  Sample
-
GA3 53.4 1.89 1.72
+
! ng/ ?L
-
GA4 67.1 1.88 2.02
+
! 260/280
-
GA5 49.5 1.92 1.86
+
! 260/230
-
GA6 84.2 1.90 2.07
+
 
-
GA7 54.6 1.94 2.08
+
|-
-
GA8 59.8 1.86 1.60
+
| GA1
-
P50 91.3 1.88 2.12
+
| 80.5
-
P51 65.8 1.88 2.03
+
| 1.88
-
_t
+
| 2.04
 +
 
 +
|-
 +
| GA2
 +
| 87.8
 +
| 1.90
 +
| 2.14
 +
 
 +
|-
 +
| GA3
 +
| 53.4
 +
| 1.89
 +
| 1.72
 +
 
 +
|-
 +
| GA4
 +
| 67.1
 +
| 1.88
 +
| 2.02
 +
 
 +
|-
 +
| GA5
 +
| 49.5
 +
| 1.92
 +
| 1.86
 +
 
 +
|-
 +
| GA6
 +
| 84.2
 +
| 1.90
 +
| 2.07
 +
 
 +
|-
 +
| GA7
 +
| 54.6
 +
| 1.94
 +
| 2.08
 +
 
 +
|-
 +
| GA8
 +
| 59.8
 +
| 1.86
 +
| 1.60
 +
 
 +
|-
 +
| P50
 +
| 91.3
 +
| 1.88
 +
| 2.12
 +
 
 +
|-
 +
| P51
 +
| 65.8
 +
| 1.88
 +
| 2.03
 +
 
 +
|-
 +
|}
Samples from the digestion reaction were run on a 1% agarose gel (shown below). From these results, it appears that none of the Gibson Assembly colonies contain the correct products.
Samples from the digestion reaction were run on a 1% agarose gel (shown below). From these results, it appears that none of the Gibson Assembly colonies contain the correct products.
   
   
-
_t Lane Sample
+
{| class = "wikitable"
-
1 2-Log DNA Ladder
+
|-
-
2 GA1
+
!  Lane
-
3 GA2
+
! Sample
-
4 GA3
+
 
-
5 GA4
+
|-
-
6 GA5
+
| 1
-
7 GA6
+
| 2-Log DNA Ladder
-
8 GA7
+
 
-
9 GA8  
+
|-
-
10 P50 Digest
+
| 2
-
11 SB1C3
+
| GA1
-
12 Undigested GA1
+
 
-
13 Undigested GA2
+
|-
-
14 Undigested GA3
+
| 3
-
15 Undigested P50
+
| GA2
-
_t
+
 
 +
|-
 +
| 4
 +
| GA3
 +
 
 +
|-
 +
| 5
 +
| GA4
 +
 
 +
|-
 +
| 6
 +
| GA5
 +
 
 +
|-
 +
| 7
 +
| GA6
 +
 
 +
|-
 +
| 8
 +
| GA7
 +
 
 +
|-
 +
| 9
 +
| GA8  
 +
 
 +
|-
 +
| 10
 +
| P50 Digest
 +
 
 +
|-
 +
| 11
 +
| SB1C3
 +
 
 +
|-
 +
| 12
 +
| Undigested GA1
 +
 
 +
|-
 +
| 13
 +
| Undigested GA2
 +
 
 +
|-
 +
| 14
 +
| Undigested GA3
 +
 
 +
|-
 +
| 15
 +
| Undigested P50
 +
 
 +
|-
 +
|}
[[File:UCB-induced cc9- gel- 140609.jpg]]
[[File:UCB-induced cc9- gel- 140609.jpg]]
   
   
-
Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. 20 plates were made with ½ containing Kan and ½ containing Amp and Kan. The plates were made with increasing amounts of ATC.
+
Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. 20 plates were made with ? containing Kan and ? containing Amp and Kan. The plates were made with increasing amounts of ATC.
gRNAs from cPEC were transformed and plated according to protocol with controls.
gRNAs from cPEC were transformed and plated according to protocol with controls.
Line 543: Line 1,309:
AmilCP restriction digestion was performed on the original and PCR amplified samples and the products run on a 1% agarose gel as shown below:
AmilCP restriction digestion was performed on the original and PCR amplified samples and the products run on a 1% agarose gel as shown below:
-
_t Reactants PCR AmilCP Original AmilCP
+
{| class = "wikitable"
-
Buffer 3 1.25 μL 1.25 μL
+
|-
-
BxsI 1.0 μL 2.5 μL
+
!  Reactants
-
DNA (125 ng) 4.0 μL 1.25 μL
+
! PCR AmilCP
-
Nuclease Free H2O 6.25 μL 7.5 μL
+
! Original AmilCP
-
Final Reaction Volume 12.5 μL 12.5 μL
+
 
-
_t
+
|-
 +
| Buffer 3
 +
| 1.25 ?L
 +
| 1.25 ?L
 +
 
 +
|-
 +
| BxsI
 +
| 1.0 ?L
 +
| 2.5 ?L
 +
 
 +
|-
 +
| DNA (125 ng)
 +
| 4.0 ?L
 +
| 1.25 ?L
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 6.25 ?L
 +
| 7.5 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 12.5 ?L
 +
| 12.5 ?L
 +
 
 +
|-
 +
|}
6/10/14
6/10/14
   
   
cPEC was performed to join the gRNA (as primer dimers) with the p51 plasmid and AmilCP.
cPEC was performed to join the gRNA (as primer dimers) with the p51 plasmid and AmilCP.
   
   
-
First, primers had to be dimerized. Master mixes of all gRNA primers were created and aliquots made by adding 5 μL of each to 40 μL nuclease free H2O (N.F. H2O). Two separate primer mixes were made (one for p51 and one for AmilCP) using 5 μL of each forward and reverse primer dimer that were added to 40 μL of N.F. H2O to produce a dilution (1:1:8). 5 μL of each primer mix was added to 25 μL of the Q5 master mix and 20 μL N.F. H2O to make a 50 μL total reaction for each sample.Products were checked by gel electrophoresis (shown below).
+
First, primers had to be dimerized. Master mixes of all gRNA primers were created and aliquots made by adding 5 ?L of each to 40 ?L nuclease free H2O (N.F. H2O). Two separate primer mixes were made (one for p51 and one for AmilCP) using 5 ?L of each forward and reverse primer dimer that were added to 40 ?L of N.F. H2O to produce a dilution (1:1:8). 5 ?L of each primer mix was added to 25 ?L of the Q5 master mix and 20 ?L N.F. H2O to make a 50 ?L total reaction for each sample.Products were checked by gel electrophoresis (shown below).
[[File:UCB-induced cc9- gel-140610.jpg]]
[[File:UCB-induced cc9- gel-140610.jpg]]
   
   
Line 562: Line 1,354:
-
_t  Reactants Volume
+
{| class = "wikitable"
-
Q5 Master Mix 25.0 μL
+
|-
-
Primer Mix 2.5 μL
+
!  Reactants
-
DNA (1:10 dilution) 1.0 μL
+
! Volume
-
Nuclease Free H2O 25.5 μL
+
 
-
Final Reaction Volume 54.0 μL
+
|-
-
  _t
+
| Q5 Master Mix
 +
| 25.0 ?L
 +
 
 +
|-
 +
| Primer Mix
 +
| 2.5 ?L
 +
 
 +
|-
 +
| DNA (1:10 dilution)
 +
| 1.0 ?L
 +
 
 +
|-
 +
| Nuclease Free H2O
 +
| 25.5 ?L
 +
 
 +
|-
 +
| Final Reaction Volume
 +
| 54.0 ?L
 +
 
 +
|-
 +
| _t
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Following the cPEC reactions, Nanodrops were taken to assess the concentration and purity of the samples:
-
Following the cPEC reactions, Nanodrops were taken to assess the concentration and purity of the samples:
+
|-
-
_t Sample ng/ μL 260/280 260/230
+
|}
P51 416.9 1.82 0.67
P51 416.9 1.82 0.67
AmilCP 430.5 1.82 0.67
AmilCP 430.5 1.82 0.67
Line 580: Line 1,398:
SC1 464.6 1.80 0.73
SC1 464.6 1.80 0.73
SC2 475.1 1.80 0.74
SC2 475.1 1.80 0.74
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
Titration Experiment: Done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. The plan is to make 50 mL of BWF+ w/cas9 and BWF+ w/o cas9 with a starting OD600 of 0.05. These will be placed in an incubator and allowed to grow until reaching 0.5. They will then be diluted again to 0.05 (in 50 mL). Serial dilutions will be made and plated on the prepared plates which will be incubated overnight.
+
|-
-
   
+
|
-
Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.
+
 
-
           
+
|-
-
_t Sample OD Amount added to 50 mL
+
| Titration Experiment: Done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. The plan is to make 50 mL of BWF+ w/cas9 and BWF+ w/o cas9 with a starting OD600 of 0.05. These will be placed in an incubator and allowed to grow until reaching 0.5. They will then be diluted again to 0.05 (in 50 mL). Serial dilutions will be made and plated on the prepared plates which will be incubated overnight.
-
BWF+ 0.544 460 μL
+
 
-
BWF+ w/cas9 0.556 450 μL
+
|-
-
_t
+
|  
 +
 
 +
|-
 +
| Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.
 +
 
 +
|-
 +
|           
 +
|
 +
 
 +
|-
 +
|}
 +
BWF+ 0.544 460 ?L
 +
BWF+ w/cas9 0.556 450 ?L
 +
{| class = "wikitable"
 +
|-
 +
! 
 +
 
 +
|-
 +
|
-
_t Time BWF+ BWF+ w/cas9
+
|-
 +
|}
T0 0.059 0.058
T0 0.059 0.058
T1 0.081 0.078
T1 0.081 0.078
Line 603: Line 1,442:
T9† 0.561 0.490
T9† 0.561 0.490
T10 0.057 0.056
T10 0.057 0.056
-
_t
+
{| class = "wikitable"
-
*At T6, the cultures were again diluted down (5.17 mL BWF+, 5.72 mL BWF+ w/cas9 into 50 mL).
+
|-
-
†After T9, 4.898 mL BWF+ and 4.278 mL BWF+ w/ cas9 were again diluted into 50 mL LB. A final OD was taken to ensure that the final OD was close to 0.05 before the cells were plated.
+
!
-
+
-
Cells were plated on the previously prepared plates containing increasing concentrations of ATC as previously described (6/9/14) and placed in 37°C incubator overnight.
+
-
+
-
6/11/14
+
|-
-
+
| *At T6, the cultures were again diluted down (5.17 mL BWF+, 5.72 mL BWF+ w/cas9 into 50 mL).
-
Titration Experiment: Results
+
-
+
-
As seen from the images in the table below, the cas9 protein inhibits growth of the cells even prior to removal of repression by the TetR repressor, demonstrating some leaky expression. Increasing ATC concentrations results in increased cas9 expression in the cells as expected.
+
 +
|-
 +
| †After T9, 4.898 mL BWF+ and 4.278 mL BWF+ w/ cas9 were again diluted into 50 mL LB. A final OD was taken to ensure that the final OD was close to 0.05 before the cells were plated.
-
[[File:UCB-induced cc9-titration plates-140611.JPG]]
+
|-
 +
| 
 +
|-
 +
| Cells were plated on the previously prepared plates containing increasing concentrations of ATC as previously described (6/9/14) and placed in 37°C incubator overnight.
-
Nanodrop of gel extracted (GE) and mini-prep (MP) products as shown below:
+
|-
-
   
+
| 
-
_t Sample ng/ μL 260/280 260/230
+
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 6/11/14
 +
 
 +
|-
 +
| 
 +
 
 +
|-
 +
|  Titration Experiment: Results
 +
 
 +
|-
 +
| 
 +
 
 +
|-
 +
| As seen from the images in the table below, the cas9 protein inhibits growth of the cells even prior to removal of repression by the TetR repressor, demonstrating some leaky expression. Increasing ATC concentrations results in increased cas9 expression in the cells as expected.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| [[File:UCB-induced cc9-titration plates-140611.JPG]]
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Nanodrop of gel extracted (GE) and mini-prep (MP) products as shown below:
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
|}
GE P51 13.7 1.94 1.18
GE P51 13.7 1.94 1.18
GE AmilCP PCR 27.3 1.81 1.19
GE AmilCP PCR 27.3 1.81 1.19
Line 645: Line 1,524:
Gel extraction of Cas9 done to protocol followed by a nanodrop (results below).
Gel extraction of Cas9 done to protocol followed by a nanodrop (results below).
            
            
-
_t Sample ng/ μL 260/280 260/230
+
{| class = "wikitable"
-
Cas9 5.5 2.12 0.59
+
|-
-
_t
+
!  Sample
-
P50 PCR amplification performed using the following amounts:
+
! ng/ ?L
-
- 25 μL Master Mix
+
! 260/280
-
- 2.5 μL of forward primer (10 μM)
+
! 260/230
-
- 2.5 μL of reverse primer (10 μM)
+
-
- 1.0 μL DNA (1:50 dilution)
+
-
- 19.0 μL NF H2O
+
-
+
-
Two samples prepared with different thermocycler settings as follows:
+
-
+
|-
-
_t 1st Round 2nd Round
+
| Cas9
 +
| 5.5
 +
| 2.12
 +
| 0.59
 +
 
 +
|-
 +
|  _t
 +
 
 +
|-
 +
| P50 PCR amplification performed using the following amounts:
 +
 
 +
|-
 +
| -
 +
| 25 ?L Master Mix
 +
 
 +
|-
 +
| -
 +
| 2.5 ?L of forward primer (10 ?M)
 +
 
 +
|-
 +
| -
 +
| 2.5 ?L of reverse primer (10 ?M)
 +
 
 +
|-
 +
| -
 +
| 1.0 ?L DNA (1:50 dilution)
 +
 
 +
|-
 +
| -
 +
| 19.0 ?L NF H2O
 +
 
 +
|-
 +
| 
 +
 
 +
|-
 +
| Two samples prepared with different thermocycler settings as follows:
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 
 +
 
 +
|-
 +
|}
Initial Denature 98°C/30 sec 98°C/2 min
Initial Denature 98°C/30 sec 98°C/2 min
Denature 98°C/30 sec 98°C/30 sec
Denature 98°C/30 sec 98°C/30 sec
Line 665: Line 1,583:
# of cycles 34 30
# of cycles 34 30
Final Extension 72°C/2 min 72°C/5 min
Final Extension 72°C/2 min 72°C/5 min
-
_t
+
{| class = "wikitable"
-
Overnight cultures prepared on GA1-GA5, 729-1, 729-2, 729-3, and 759-1, 759-2
+
|-
-
Colonies taken from SC1 plate for overnight cultures (SC-1, SC-2) with 5 mL LB, 10 μL Amp (50 ng/mL)
+
!
-
6/13/14
+
|-
-
   
+
| Overnight cultures prepared on GA1-GA5, 729-1, 729-2, 729-3, and 759-1, 759-2
-
Mini-prep done on O/N cultures SC1-1 and SC1-2 followed by a nanodrop. Remainder of culture was spun down into a pellet and frozen.
+
 
-
   
+
|-
-
_t Sample ng/ μL 260/280 260/230
+
| Colonies taken from SC1 plate for overnight cultures (SC-1, SC-2) with 5 mL LB, 10 ?L Amp (50 ng/mL)
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 6/13/14
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
| Mini-prep done on O/N cultures SC1-1 and SC1-2 followed by a nanodrop. Remainder of culture was spun down into a pellet and frozen.
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
|}
SC1-1 30.7 2.02 2.06
SC1-1 30.7 2.02 2.06
SC1-2 32.5 1.94 1.48
SC1-2 32.5 1.94 1.48
Line 680: Line 1,616:
Restriction Digestion done following manufacturer’s protocols on the samples as listed below. All were incubated at 37°C for 1 hour followed by 20 minute heat inactivation at 80°C.
Restriction Digestion done following manufacturer’s protocols on the samples as listed below. All were incubated at 37°C for 1 hour followed by 20 minute heat inactivation at 80°C.
   
   
-
_t Sample Buffer 3 10X BSA Enzyme 1 Enzyme 2 Enzyme Mix* DNA (200 ng) MQ H2O Total Volume
+
{| class = "wikitable"
-
756-1 2.0 μL 2.0 μL 0.5 μL EagI 0.5 μL XbaI -- 1.0 μL 14.0 μL 20.0 μL
+
|-
-
756-2 2.0 μL 2.0 μL 0.5 μL EagI 0.5 μL XbaI -- 1.87 μL 13.13 μL 20.0 μL
+
!  Sample
-
GA1 2.0 μL 2.0 μL -- -- 1.0 μL 1.85 μL 13.15 μL 20.0 μL
+
! Buffer 3
-
GA2 2.0 μL 2.0 μL -- -- 1.0 μL 1.38 μL 13.62 μL 20.0 μL
+
! 10X BSA
-
GA3 2.0 μL 2.0 μL -- -- 1.0 μL 1.19 μL 13.81 μL 20.0 μL
+
! Enzyme 1
-
GA4 2.0 μL 2.0 μL -- -- 1.0 μL 1.53 μL 13.47 μL 20.0 μL
+
! Enzyme 2
-
GA5 2.0 μL 2.0 μL -- -- 1.0 μL 0.64 μL 14.36 μL 20.0 μL
+
! Enzyme Mix*
-
P51 2.0 μL 2.0 μL -- -- 1.0 μL 4.0 μL 11.0 μL 20.0 μL
+
! DNA (200 ng)
-
Cas9 2.0 μL 2.0 μL -- -- 1.0 μL 2.0 μL 13.0 μL 20.0 μL
+
! MQ H2O
-
_t
+
! Total Volume
 +
 
 +
|-
 +
| 756-1
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| 0.5 ?L EagI
 +
| 0.5 ?L XbaI
 +
| --
 +
| 1.0 ?L
 +
| 14.0 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| 756-2
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| 0.5 ?L EagI
 +
| 0.5 ?L XbaI
 +
| --
 +
| 1.87 ?L
 +
| 13.13 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| GA1
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 1.85 ?L
 +
| 13.15 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| GA2
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 1.38 ?L
 +
| 13.62 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| GA3
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 1.19 ?L
 +
| 13.81 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| GA4
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 1.53 ?L
 +
| 13.47 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| GA5
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 0.64 ?L
 +
| 14.36 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| P51
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 4.0 ?L
 +
| 11.0 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| Cas9
 +
| 2.0 ?L
 +
| 2.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 13.0 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
|}
*Enzyme Mix is a 1:1:23 dilution of EcoRI and BamHI
*Enzyme Mix is a 1:1:23 dilution of EcoRI and BamHI
   
   
Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
-
_t Lane Sample
+
{| class = "wikitable"
-
1 2-Log DNA Ladder
+
|-
-
2 756-1
+
!  Lane
-
3 756-2
+
! Sample
-
4 P51 Digest
+
 
-
5 Sb1C3
+
|-
-
6 GA1
+
| 1
-
7 GA2
+
| 2-Log DNA Ladder
-
8 GA3
+
 
-
9 GA4
+
|-
-
10 GA5
+
| 2
-
11 Cas9 Digest
+
| 756-1
-
_t
+
 
 +
|-
 +
| 3
 +
| 756-2
 +
 
 +
|-
 +
| 4
 +
| P51 Digest
 +
 
 +
|-
 +
| 5
 +
| Sb1C3
 +
 
 +
|-
 +
| 6
 +
| GA1
 +
 
 +
|-
 +
| 7
 +
| GA2
 +
 
 +
|-
 +
| 8
 +
| GA3
 +
 
 +
|-
 +
| 9
 +
| GA4
 +
 
 +
|-
 +
| 10
 +
| GA5
 +
 
 +
|-
 +
| 11
 +
| Cas9 Digest
 +
 
 +
|-
 +
|}
  [[File:UCB-induced cc9-gel-140613.JPG]]
  [[File:UCB-induced cc9-gel-140613.JPG]]
-
From the above gel, it does not appear that are products are as expected. A repeat digest is done of GA4 and P50. These new products were run on a 1% agarose gel with reagents as comparisons (5 μL sample plus 1 μL loading dye for each) as shown below:
+
From the above gel, it does not appear that are products are as expected. A repeat digest is done of GA4 and P50. These new products were run on a 1% agarose gel with reagents as comparisons (5 ?L sample plus 1 ?L loading dye for each) as shown below:
-
_t Lane Sample
+
{| class = "wikitable"
-
1 2-Log DNA Ladder
+
|-
-
2 GA4
+
!  Lane
-
3 P50 Digest
+
! Sample
-
4 Sb1C3
+
 
-
5 CmR1
+
|-
-
6 Cas9 PCR
+
| 1
-
7 Cas9 GE
+
| 2-Log DNA Ladder
-
8 GA4 MP
+
 
-
_t
+
|-
 +
| 2
 +
| GA4
 +
 
 +
|-
 +
| 3
 +
| P50 Digest
 +
 
 +
|-
 +
| 4
 +
| Sb1C3
 +
 
 +
|-
 +
| 5
 +
| CmR1
 +
 
 +
|-
 +
| 6
 +
| Cas9 PCR
 +
 
 +
|-
 +
| 7
 +
| Cas9 GE
 +
 
 +
|-
 +
| 8
 +
| GA4 MP
 +
 
 +
|-
 +
|}
[[File:UCB-induced cc9-gel-02-140613.JPG]]
[[File:UCB-induced cc9-gel-02-140613.JPG]]
Line 727: Line 1,832:
   
   
   
   
-
Gibson Assembly performed both with and without 2% DMSO. Each 20 μL reaction contained 10 μL Gibson Master Mix with the following as listed:   
+
Gibson Assembly performed both with and without 2% DMSO. Each 20 ?L reaction contained 10 ?L Gibson Master Mix with the following as listed:   
Reaction #1           
Reaction #1           
-
- 6.5 μL GE Cas9
+
- 6.5 ?L GE Cas9
-
- 3.5 μL GE AmilCP
+
- 3.5 ?L GE AmilCP
   
   
Reaction #2
Reaction #2
-
- 1 μL P50 PCR Product (1:10)
+
- 1 ?L P50 PCR Product (1:10)
-
- 5 μL GE AmilCP
+
- 5 ?L GE AmilCP
-
- 4 μL MQ H2O
+
- 4 ?L MQ H2O
   
   
Reaction #3
Reaction #3
-
- 1 μL P50 PCR Product (1:10)
+
- 1 ?L P50 PCR Product (1:10)
-
- 5 μL GE AmilCP
+
- 5 ?L GE AmilCP
-
- 4 μL 1:10 dilution of 2% DMSO
+
- 4 ?L 1:10 dilution of 2% DMSO
Bacterial Transformation: Done according to protocol
Bacterial Transformation: Done according to protocol
-
- GA1-GA3 diluted 1:4 transformed with 2 μL (100 μL each)
+
- GA1-GA3 diluted 1:4 transformed with 2 ?L (100 ?L each)
-
- BWF+ w/Cas9 from S.B. (25 μL full strength and 25 μL 1:10 dilution)
+
- BWF+ w/Cas9 from S.B. (25 ?L full strength and 25 ?L 1:10 dilution)
-
- BWF+ (25 μL full strength and 25 μL 1:10 dilution)
+
- BWF+ (25 ?L full strength and 25 ?L 1:10 dilution)
-
- NEB competent cells with P51 (50 μL)
+
- NEB competent cells with P51 (50 ?L)
-
- Competent cells with P51 (50 μL) for comparison
+
- Competent cells with P51 (50 ?L) for comparison
   
   
Week 6
Week 6
Line 752: Line 1,857:
6/15/14
6/15/14
   
   
-
GA colonies picked for overnight cultures from 2 plates (11 total- 3 from Plate 1, 8 from plate 2), placed in 5 mL LB with 170 ng/ μL Amp.
+
GA colonies picked for overnight cultures from 2 plates (11 total- 3 from Plate 1, 8 from plate 2), placed in 5 mL LB with 170 ng/ ?L Amp.
   
   
Line 759: Line 1,864:
Mini-prep was done to protocol on all overnight cultures followed by a nanodrop with results listed below:
Mini-prep was done to protocol on all overnight cultures followed by a nanodrop with results listed below:
   
   
-
_t Sample ng/ μL 260/280 260/230
+
{| class = "wikitable"
-
1-1 17.5 2.08 1.94
+
|-
-
1-2 54.1 1.93 1.96
+
!  Sample
-
1-3 30.4 2.00 1.78
+
! ng/ ?L
-
2-1 45.8 1.93 1.90
+
! 260/280
-
2-2 45.4 1.92 1.89
+
! 260/230
-
2-3 46.9 1.93 1.97
+
-
2-4 53.5 1.96 2.01
+
-
2-5 53.5 1.94 2.04
+
-
2-6 41.9 1.95 1.84
+
-
2-7 59.3 1.94 1.99
+
-
2-8 46.5 1.94 1.98
+
-
_t
+
-
Next, a restriction digest was performed on all the samples listed above. EcoRV was used in a 1:100 dilution with Buffer D (both from Promega). Samples were incubated for 1 hour at 37°C and heat inactivation performed for 5 minutes at 80°C. The following amounts were used for all the samples except 1-1 (DNA increased to 6.0 μL and MQ H2O was not added).
+
|-
-
- 1.0 μL Buffer D
+
| 1-1
-
- 3.0 μL Enzyme
+
| 17.5
-
- 3.0 μL sample DNA
+
| 2.08
-
- 3.0 μL MQ H2O
+
| 1.94
-
- Total Reaction Volume = 10 μL
+
-
Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
+
|-
-
_t Lane Sample
+
| 1-2
 +
| 54.1
 +
| 1.93
 +
| 1.96
 +
 
 +
|-
 +
| 1-3
 +
| 30.4
 +
| 2.00
 +
| 1.78
 +
 
 +
|-
 +
| 2-1
 +
| 45.8
 +
| 1.93
 +
| 1.90
 +
 
 +
|-
 +
| 2-2
 +
| 45.4
 +
| 1.92
 +
| 1.89
 +
 
 +
|-
 +
| 2-3
 +
| 46.9
 +
| 1.93
 +
| 1.97
 +
 
 +
|-
 +
| 2-4
 +
| 53.5
 +
| 1.96
 +
| 2.01
 +
 
 +
|-
 +
| 2-5
 +
| 53.5
 +
| 1.94
 +
| 2.04
 +
 
 +
|-
 +
| 2-6
 +
| 41.9
 +
| 1.95
 +
| 1.84
 +
 
 +
|-
 +
| 2-7
 +
| 59.3
 +
| 1.94
 +
| 1.99
 +
 
 +
|-
 +
| 2-8
 +
| 46.5
 +
| 1.94
 +
| 1.98
 +
 
 +
|-
 +
|  _t
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Next, a restriction digest was performed on all the samples listed above. EcoRV was used in a 1:100 dilution with Buffer D (both from Promega). Samples were incubated for 1 hour at 37°C and heat inactivation performed for 5 minutes at 80°C. The following amounts were used for all the samples except 1-1 (DNA increased to 6.0 ?L and MQ H2O was not added).
 +
 
 +
|-
 +
| -
 +
| 1.0 ?L Buffer D
 +
 
 +
|-
 +
| -
 +
| 3.0 ?L Enzyme
 +
 
 +
|-
 +
| -
 +
| 3.0 ?L sample DNA
 +
 
 +
|-
 +
| -
 +
| 3.0 ?L MQ H2O
 +
 
 +
|-
 +
| -
 +
| Total Reaction Volume = 10 ?L
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
 +
 
 +
|-
 +
|}
1 2-Log DNA Ladder
1 2-Log DNA Ladder
2 1-1
2 1-1
Line 794: Line 1,986:
11 2-7
11 2-7
12 2-8
12 2-8
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
Repeated digestion (protocol above) on select samples to run again for more definition on gel. Samples are run on a 1% agarose gel. The following lists the sample loaded into each lane.
+
|-
 +
|
-
_t Lane Sample
+
|-
 +
| Repeated digestion (protocol above) on select samples to run again for more definition on gel. Samples are run on a 1% agarose gel. The following lists the sample loaded into each lane.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
1 2-Log DNA Ladder
1 2-Log DNA Ladder
2 1-1
2 1-1
Line 807: Line 2,009:
7 Digested p50
7 Digested p50
8 2-Log DNA Ladder
8 2-Log DNA Ladder
-
_t
+
{| class = "wikitable"
-
[[File:UCB-induced cc9-gel-140616.JPG]]
+
|-
 +
!
-
Bacterial Transformation: Done according to protocol
+
|-
-
+
| [[File:UCB-induced cc9-gel-140616.JPG]]
-
- 7 CPEC products including p51 as the negative control (10 μL  1:4 dilution) with 40 μL  DH5α
+
-
- BWF+ w (40μL)/ S.B. cas9 (1 μL)
+
-
- BWF+ (1 μL)
+
-
Gibson Assembly products 1-1 thru 1-8 and 2-1 thru 2-3 pelleted down and frozen 
+
|-
 +
|
 +
|-
 +
| Bacterial Transformation: Done according to protocol
-
6/17/14
+
|-
 +
|
 +
|
-
_t Sample Buffer Enzyme 1 EnzymeMix* DNA (200 ng) MQ H2O Total Volume
+
|-
-
GA x 1-1 1.0 μL cutsmart 3.0 μL XbaI (1:50) -- 3.0 μL 3.0 μL 10.0 μL
+
| -
-
GA x 1-2 1.0 μL cutsmart 3.0 μL XbaI (1:50) --     3.0  μL 3.0 μL 10.0 μL
+
| 7 CPEC products including p51 as the negative control (10 ?L  1:4 dilution) with 40 ?L DH5?
-
GA x 2-1 1.0 μL cutsmart 3.0 μL XbaI (1:50) -- 3.0 μL 3.0 μL 10.0 μL
+
-
p50 linear 1.0 μL cutsmart 3.0 μL SepI (1:50) -- 3.0 μL 3.0 μL 10.0 μL
+
-
GA H 1-1 1.0 μL Fast green digest -- 3.0 μL 3.0 μL 3.0 μL 10.0 μL
+
-
GA H 1-2 1.0 μL Fast green digest -- 3.0 μL 3.0 μL 3.0 μL 10.0 μL
+
-
GA H 2-1 1.0 μL Fast green digest -- 3.0 μL 3.0 μL 3.0 μL 10.0 μL
+
-
p50 1.0 μL Fast green digest -- 3.0 μL 3.0 μL 3.0 μL 10.0 μL
+
-
_t
+
-
*Enzyme mix: 1:1:23 dilution of xhoI and HindIII
+
-
Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
+
|-
-
_t Lane Sample
+
| -
 +
| BWF+ w (40?L)/ S.B. cas9 (1 ?L)
 +
 
 +
|-
 +
| -
 +
| BWF+ (1 ?L)
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Gibson Assembly products 1-1 thru 1-8 and 2-1 thru 2-3 pelleted down and frozen 
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 6/17/14
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
 +
GA x 1-1 1.0 ?L cutsmart 3.0 ?L XbaI (1:50) -- 3.0 ?L 3.0 ?L 10.0 ?L
 +
GA x 1-2 1.0 ?L cutsmart 3.0 ?L XbaI (1:50) --     3.0  ?L 3.0 ?L 10.0 ?L
 +
GA x 2-1 1.0 ?L cutsmart 3.0 ?L XbaI (1:50) -- 3.0 ?L 3.0 ?L 10.0 ?L
 +
p50 linear 1.0 ?L cutsmart 3.0 ?L SepI (1:50) -- 3.0 ?L 3.0 ?L 10.0 ?L
 +
GA H 1-1 1.0 ?L Fast green digest -- 3.0 ?L 3.0 ?L 3.0 ?L 10.0 ?L
 +
GA H 1-2 1.0 ?L Fast green digest -- 3.0 ?L 3.0 ?L 3.0 ?L 10.0 ?L
 +
GA H 2-1 1.0 ?L Fast green digest -- 3.0 ?L 3.0 ?L 3.0 ?L 10.0 ?L
 +
p50 1.0  ?L Fast green digest -- 3.0 ?L 3.0 ?L 3.0 ?L 10.0 ?L
 +
{| class = "wikitable"
 +
|-
 +
!
 +
!
 +
 
 +
|-
 +
| *Enzyme mix: 1:1:23 dilution of xhoI and HindIII
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
 +
 
 +
|-
 +
|}
1 2-Log DNA Ladder
1 2-Log DNA Ladder
2 GA x 1-1
2 GA x 1-1
Line 847: Line 2,094:
11 2-Log DNA Ladder
11 2-Log DNA Ladder
-
_t
+
{| class = "wikitable"
-
[[File:UCB-induced cc9-gel-140617.JPG]]
+
|-
 +
!
-
6/18/14
+
|-
-
   
+
|  [[File:UCB-induced cc9-gel-140617.JPG]]
-
Colony PCR is used to rapidly screen colonies from cPEC reactions. A colony is pricked using a pipette tip, avoiding scooping up any LB agar since this inhibits the PCR reaction. There are ~10^9 cells are in each colony - just barely touching the colony and transferring a tiny fraction of it is more than sufficient for this reaction. This tip is rubbed into the PCR tube . Tips  were then ejected into  3ml LB+AMP  for O/N culture.The following was added to each tube to make a 25 μL reaction.
+
 
-
- 12.5 μL 2X Master Mix
+
|-
-
- 1.25 μL of forward primer (10 μM)
+
|
-
- 1.25 μL of reverse primer (10 μM)
+
 
-
- 10.0 μL MQ H2O
+
|-
-
   
+
| 6/18/14
-
For this reaction, the thermocycler was programmed as follows (note the longer time to initially lyse the cells and denature the DNA).
+
 
-
   
+
|-
-
_t Initial Denature 98°C/10 min
+
|  
 +
 
 +
|-
 +
| Colony PCR is used to rapidly screen colonies from cPEC reactions. A colony is pricked using a pipette tip, avoiding scooping up any LB agar since this inhibits the PCR reaction. There are ~10^9 cells are in each colony - just barely touching the colony and transferring a tiny fraction of it is more than sufficient for this reaction. This tip is rubbed into the PCR tube . Tips  were then ejected into  3ml LB+AMP  for O/N culture.The following was added to each tube to make a 25 ?L reaction.
 +
 
 +
|-
 +
| -
 +
| 12.5 ?L 2X Master Mix
 +
 
 +
|-
 +
| -
 +
| 1.25 ?L of forward primer (10 ?M)
 +
 
 +
|-
 +
| -
 +
| 1.25 ?L of reverse primer (10 ?M)
 +
 
 +
|-
 +
| -
 +
| 10.0 ?L MQ H2O
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
| For this reaction, the thermocycler was programmed as follows (note the longer time to initially lyse the cells and denature the DNA).
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
|}
Denature 98°C/30 sec
Denature 98°C/30 sec
Anneal 65°C/30 sec
Anneal 65°C/30 sec
Line 866: Line 2,145:
# of cycles 35
# of cycles 35
Final Extension 72°C/5 min
Final Extension 72°C/5 min
-
_t
+
{| class = "wikitable"
-
6/19/14
+
|-
-
Glycerol stocks done to protocol for two colonies from each gRNA that were indicated positive by colony PCR results.
+
! 
-
Week 7
+
|-
 +
| 6/19/14
-
6/23/14
+
|-
-
Chemically competent cells of SBcas9 • BWF+. Done according to protocol.
+
| Glycerol stocks done to protocol for two colonies from each gRNA that were indicated positive by colony PCR results.  
-
6/25/14
+
|-
-
Mini prep done to protocol on CPEC products.
+
|
-
Nanodrops were taken to assess the concentration and purity of the samples:
+
|-
-
_t Sample ng/ μL 260/280 260/230
+
| Week 7
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 6/23/14
 +
 
 +
|-
 +
| Chemically competent cells of SBcas9 • BWF+. Done according to protocol.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 6/25/14
 +
 
 +
|-
 +
| Mini prep done to protocol on CPEC products.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Nanodrops were taken to assess the concentration and purity of the samples:
 +
 
 +
|-
 +
|}
2H 65.2 1.88 2.21
2H 65.2 1.88 2.21
     3E 56.3 1.91 2.01
     3E 56.3 1.91 2.01
Line 891: Line 2,198:
11B 43.9 1.86 1.33
11B 43.9 1.86 1.33
12B 57.5 1.83 1.53
12B 57.5 1.83 1.53
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
Sequencing 12 CPEC products: 12.5 μL DNA + 2.5 μL  10μm primer (CRE22)
+
|-
-
+
|
-
_t CPEC product
+
|-
 +
| Sequencing 12 CPEC products: 12.5 ?L DNA + 2.5 ?L  10?m primer (CRE22)
 +
 
 +
|-
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
1 1A
1 1A
2 1C
2 1C
Line 909: Line 2,233:
11 6C
11 6C
12 6F
12 6F
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 6/26/14
 +
 
 +
|-
 +
| Sequencing: Total volume 0f 15 ?L
 +
 
 +
|-
 +
|}
 +
1 2B 12.5?L 2.5 ?L --
 +
2 2C 12.0?L 2.5?L 0.5?L
 +
3 3B 10.3?L 2.5?L 2.2?L
 +
4 3H 11?L 2.5?L 1.5?L
 +
5 4E 12.5?L 2.5?L --
 +
6 5E 11.3?L 2.5?L 1.2?L
 +
7 11B 12.5?L 2.5?L --
 +
8 2H 8?L 2.5?L 4.5?L
 +
9 6B 8?L 2.5?L 4.5?L
 +
10 7B 8?L 2.5?L 4.5?L
 +
11 8B 8?L 2.5?L 4.5?L
 +
12 9B 8?L 2.5?L 4.5?L
 +
13 4C 8?L 2.5?L 4.5?L
 +
14 3E 10?L 2.5?L 2.5?L
 +
15 5B 10?L 2.5?L 2.5?L
 +
16 10C 10?L 2.5?L 2.5?L
 +
17 12B 10?L 2.5?L 2.5?L
 +
 
 +
{| class = "wikitable"
 +
|-
 +
!
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Bacterial Transformation of SBcas9• ER: Done according  to protocol.
 +
 
 +
|-
 +
|
-
6/26/14  
+
|-
-
Sequencing: Total volume 0f 15 μL
+
| 6/27/14
-
_t CPEC Product DNA μL Primer CRE22 MQ H2O
+
-
1 2B 12.5μL 2.5 μL --
+
-
2 2C 12.0μL 2.5μL 0.5μL
+
-
3 3B 10.3μL 2.5μL 2.2μL
+
-
4 3H 11μL 2.5μL 1.5μL
+
-
5 4E 12.5μL 2.5μL --
+
-
6 5E 11.3μL 2.5μL 1.2μL
+
-
7 11B 12.5μL 2.5μL --
+
-
8 2H 8μL 2.5μL 4.5μL
+
-
9 6B 8μL 2.5μL 4.5μL
+
-
10 7B 8μL 2.5μL 4.5μL
+
-
11 8B 8μL 2.5μL 4.5μL
+
-
12 9B 8μL 2.5μL 4.5μL
+
-
13 4C 8μL 2.5μL 4.5μL
+
-
14 3E 10μL 2.5μL 2.5μL
+
-
15 5B 10μL 2.5μL 2.5μL
+
-
16 10C 10μL 2.5μL 2.5μL
+
-
17 12B 10μL 2.5μL 2.5μL
+
-
_t
+
|-
 +
| Mini prep done to protocol on CPEC products.
-
Bacterial Transformation of SBcas9• ER: Done according  to protocol.
+
|-
 +
| Nanodrops were taken to assess the concentration and purity of the samples:
-
6/27/14
+
|-
-
Mini prep done to protocol on CPEC products.
+
|}
-
Nanodrops were taken to assess the concentration and purity of the samples:
+
-
_t Sample ng/ μL 260/280 260/230
+
1A 66.9 1.90 2.26
1A 66.9 1.90 2.26
     1C 61.3 1.92 2.20
     1C 61.3 1.92 2.20
Line 949: Line 2,299:
11B 40.5 1.94 2.01
11B 40.5 1.94 2.01
12B 51.9 1.89 1.17
12B 51.9 1.89 1.17
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
Sequenced all of the above  products (total volume of 15 μL: 10 μL DNA+ 2.5 μLprimer (CRE22)+2 .5 μL  MQ H2O
+
|-
-
Exceptions:
+
|
-
-9B received 12.5 μL DNA + 2.5 μL primer (CRE22)
+
-
-12B received p51 seq1 as the primer 
+
-
Glycerol stock of all the above samples to protocol: 1:1 w/ 40% glycerol for a total volume of 500 μL each.  
+
|-
 +
| Sequenced all of the above  products (total volume of 15 ?L: 10 ?L DNA+ 2.5 ?Lprimer (CRE22)+2 .5 ?L  MQ H2O
-
Positive sequencing results: 1A, 1C, 2B, 3B, 3H, 9B, 10C, 11B, 12B 
+
|-
 +
| Exceptions:  
-
Bacterial Transformation of SBcas9• ER and PUC19 as the positive control: Done according to protocol.
+
|-
 +
| -9B received 12.5 ?L DNA + 2.5 ?L primer (CRE22)
-
Prepared 5mL O/N cultures in LB+AMP of colonies selected from glycerol stocks
+
|-
-
   
+
| -12B received p51 seq1 as the primer 
-
Mini preps done to protocol on the below samples
+
 
-
Nanodrops were taken to assess the concentration and purity of the samples:
+
|-
-
_t Sample ng/ μL 260/280 260/230
+
|
 +
 
 +
|-
 +
| Glycerol stock of all the above samples  to protocol: 1:1 w/ 40% glycerol for a total volume of 500 ?L each.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Positive sequencing results: 1A, 1C, 2B, 3B, 3H, 9B, 10C, 11B, 12B 
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Bacterial Transformation of SBcas9• ER and PUC19 as the positive control: Done according to protocol.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Prepared 5mL O/N cultures in LB+AMP of colonies selected from glycerol stocks
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
| Mini preps done to protocol on the below samples
 +
 
 +
|-
 +
| Nanodrops were taken to assess the concentration and purity of the samples:
 +
 
 +
|-
 +
|}
1A 78.7 1.90 2.08
1A 78.7 1.90 2.08
     1C 54.3 1.88 1.76
     1C 54.3 1.88 1.76
Line 972: Line 2,358:
3B 52.1 1.91 1.96
3B 52.1 1.91 1.96
3H 59.7 1.89 2.03
3H 59.7 1.89 2.03
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
Week 8
+
|-
 +
|
-
6/30/14
+
|-
-
Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51: Done according to protocol.
+
| Week 8
-
7/1/14
+
|-
 +
|
-
Nanodrops were taken to assess the concentration of the samples to use in a cPEC reaction.  
+
|-
-
_t Sample ng/ μL
+
| 6/30/14
 +
 
 +
|-
 +
| Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51: Done according to protocol.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 7/1/14
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Nanodrops were taken to assess the concentration of the samples to use in a cPEC reaction.  
 +
 
 +
|-
 +
|}
p50 Amplicon 27.3
p50 Amplicon 27.3
     GE AmilCP 493.9
     GE AmilCP 493.9
-
_t
+
{| class = "wikitable"
-
*Gel extracted
+
|-
 +
!
 +
!
 +
!
 +
!
 +
!
 +
!
 +
!
 +
 
 +
|-
 +
| *Gel extracted
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| cPEC performed to combine the p50 amplicon with AmilCP following the cPEC protocol and using the following amounts:
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
 +
Q5 Master Mix 12.5 ?L
 +
Insert (AmilCP) 5 ?L
 +
Template DNA p50 2.0 ?L (1:20 dilution)
 +
25% DMSO 2.0 ?L
 +
Nuclease Free H2O 3.5 ?L
 +
Final Reaction Volume 25.0 ?L
 +
{| class = "wikitable"
 +
|-
 +
!
-
cPEC performed to combine the p50 amplicon with AmilCP following the cPEC protocol and using the following amounts:
+
|-
 +
|
-
_t Reactants Volume
+
|-
-
Q5 Master Mix 12.5 μL
+
| Thermocycler conditions:
-
Insert (AmilCP) 5 μL
+
-
Template DNA p50 2.0 μL (1:20 dilution)
+
-
25% DMSO 2.0 μL
+
-
Nuclease Free H2O 3.5 μL
+
-
Final Reaction Volume 25.0 μL
+
-
_t
+
-
Thermocycler conditions:
+
|-
 +
|
-
_t cPEC
+
|-
 +
|}
Initial Denature 98°C/30 sec
Initial Denature 98°C/30 sec
Denature 98°C/10 sec
Denature 98°C/10 sec
Line 1,008: Line 2,444:
# of cycles 10
# of cycles 10
Final Extension 72°C/10 min
Final Extension 72°C/10 min
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
Successful Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51 (from 6/30) . O/N cultures of each were then made.
+
|-
 +
|
-
7/4/14
+
|-
-
Electroporation of p50 amplicon • AmilCP with Marcello  
+
| Successful Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51 (from 6/30) . O/N cultures of each were then made.
-
7/7/14  
+
 
-
Electroporation was unsuccessful. Redone using Bacterial Transformation done to protocol.  
+
|-
-
Primer design for qPCR  
+
|
-
Streaked p50 on plate  
+
 
-
Week 9
+
|-
-
7/8/14
+
| 7/4/14
-
mini prepped p50 done to protocol  
+
 
-
Colony PCR done to protocol on the p50+AmilCP transformation product . Selected 12 colonies.
+
|-
-
*PCR tube lids opened in thermocycler for samples: 8, 12, and positive control  
+
| Electroporation of p50 amplicon • AmilCP with Marcello  
-
Colony PCR products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
+
 
-
_t Lane Sample
+
|-
 +
| 7/7/14  
 +
 
 +
|-
 +
| Electroporation was unsuccessful. Redone using Bacterial Transformation done to protocol.  
 +
 
 +
|-
 +
| Primer design for qPCR  
 +
 
 +
|-
 +
| Streaked p50 on plate  
 +
 
 +
|-
 +
| Week 9
 +
 
 +
|-
 +
| 7/8/14
 +
 
 +
|-
 +
| mini prepped p50 done to protocol  
 +
 
 +
|-
 +
| Colony PCR done to protocol on the p50+AmilCP transformation product . Selected 12 colonies.
 +
 
 +
|-
 +
|
 +
| *PCR tube lids opened in thermocycler for samples: 8, 12, and positive control  
 +
 
 +
|-
 +
| Colony PCR products are run on a 1% agarose gel. The following lists the sample loaded into each lane.
 +
 
 +
|-
 +
|}
1 2-Log DNA Ladder
1 2-Log DNA Ladder
2 1
2 1
Line 1,040: Line 2,511:
14 (+) control
14 (+) control
15 (-) control
15 (-) control
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
7/9/14
+
|-
-
Mini prepped colony PCR samples: 1,7,8,10,11, and p50
+
|
-
Followed by nanodrop with results listed below:
+
 
-
   
+
|-
-
_t Sample ng/ μL 260/280 260/230
+
| 7/9/14
 +
 
 +
|-
 +
| Mini prepped colony PCR samples: 1,7,8,10,11, and p50
 +
 
 +
|-
 +
| Followed by nanodrop with results listed below:
 +
 
 +
|-
 +
|  
 +
 
 +
|-
 +
|}
1 89 1.90 2.22
1 89 1.90 2.22
7 110.9 1.88 2.22
7 110.9 1.88 2.22
Line 1,053: Line 2,538:
11 93.6 1.89 2.21
11 93.6 1.89 2.21
p50 191.7 1.87 2.35
p50 191.7 1.87 2.35
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
The above samples (except 8) were sent for sequencing.
+
|-
-
Total volume 0f 15 μL
+
|
-
_t Product DNA μL Primer CRE22 MQ H2O
+
-
1 1 6.0μL 5.0 μL 4.0μL
+
-
2 7 5.0μL 5.0μL 5.0μL
+
-
3 10 4.0μL 5.0μL 6.0μL
+
-
4 11 6.0μL 5.0μL 4.0μL
+
-
_t
+
-
Transformation done to protocol on all gRNAs into BWF+
+
|-
 +
| The above samples (except 8) were sent for sequencing.
-
7/10/14
+
|-
-
Site directed Mutagenesis (SDM) performed on original p50, 1, 7, 10, 11 to remove Xba1 from p50
+
| Total volume 0f 15 ?L
-
Primer names: p50TetRXba1F and  p50TetRXba1R
+
-
Primer Mix: 1 μL forward + 1 μL reverse + 18 μL MQ H2O
+
-
PCR run on the following samples of p50: p50 DNA dilution for original, 1,7,10, and 11
+
|-
-
p50 DNA sample original- 1:38 dilution
+
|}
-
p50 DNA sample 1- 1:18 dilution
+
1 1 6.0?L 5.0 ?L 4.0?L
-
p50 DNA sample 7- 1:22 dilution
+
2 7 5.0?L 5.0?L 5.0?L
-
p50 DNA sample 10- 1:28 dilution
+
3 10 4.0?L 5.0?L 6.0?L
-
p50 DNA sample 11- 1:19 dilution
+
4 11 6.0?L 5.0?L 4.0?L
 +
{| class = "wikitable"
 +
|-
 +
!
 +
|-
 +
|
 +
|-
 +
| Transformation done to protocol on all gRNAs into BWF+
-
_t Reactants Volume
+
|-
-
Q5 Master Mix 12.5 μL
+
|
-
10 μM primer mix 2.5 μL
+
-
DNA p50 2.0 μL
+
-
Nuclease Free H2O 8.0 μL
+
-
Final Reaction Volume 25.0 μL
+
-
_t
+
-
Thermocycler conditions:
+
|-
 +
| 7/10/14
-
_t
+
|-
 +
| Site directed Mutagenesis (SDM) performed on original p50, 1, 7, 10, 11 to remove Xba1 from p50
 +
 
 +
|-
 +
| Primer names: p50TetRXba1F and  p50TetRXba1R
 +
 
 +
|-
 +
| Primer Mix: 1 ?L forward + 1 ?L reverse + 18 ?L MQ H2O
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| PCR run on the following samples of p50: p50 DNA dilution for original, 1,7,10, and 11
 +
 
 +
|-
 +
| p50 DNA sample original- 1:38 dilution
 +
 
 +
|-
 +
| p50 DNA sample 1- 1:18 dilution
 +
 
 +
|-
 +
| p50 DNA sample 7- 1:22 dilution
 +
 
 +
|-
 +
| p50 DNA sample 10- 1:28 dilution
 +
 
 +
|-
 +
| p50 DNA sample 11- 1:19 dilution
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
 +
Q5 Master Mix 12.5 ?L
 +
10 ?M primer mix 2.5 ?L
 +
DNA p50 2.0 ?L
 +
Nuclease Free H2O 8.0 ?L
 +
Final Reaction Volume 25.0 ?L
 +
{| class = "wikitable"
 +
|-
 +
!
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Thermocycler conditions:
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
Initial Denature 98°C/30 sec
Initial Denature 98°C/30 sec
Denature 98°C/30 sec
Denature 98°C/30 sec
Line 1,097: Line 2,640:
# of cycles 15 cycles
# of cycles 15 cycles
Final Extension 72°C/5 minutes
Final Extension 72°C/5 minutes
-
_t
+
{| class = "wikitable"
-
SDM performed on 6 gRNAs and the original p51 to remove spe1 site
+
|-
 +
!
-
PCR preformed:
+
|-
-
_t Reactants Volume
+
| SDM performed on 6 gRNAs and the original p51 to remove spe1 site
-
Q5 Master Mix 12.5 μL
+
-
10 μM primer mix 2.5 μL
+
-
DNA p51 2.0 μL
+
-
Nuclease Free H2O 8.0 μL
+
-
Final Reaction Volume 25.0 μL
+
-
_t
+
-
*primer mix: 1 μL forward primer+ 1 μL reverse primer +18 μL MQH2O. Primers varied for each gRNA
+
-
*control consisted of DNA only, no primers
+
-
Thermocycler conditions:
+
-
_t
+
|-
 +
|
 +
 
 +
|-
 +
|
 +
|
 +
|
 +
| PCR preformed:
 +
 
 +
|-
 +
|}
 +
Q5 Master Mix 12.5 ?L
 +
10 ?M primer mix 2.5 ?L
 +
DNA p51 2.0 ?L
 +
Nuclease Free H2O 8.0 ?L
 +
Final Reaction Volume 25.0 ?L
 +
{| class = "wikitable"
 +
|-
 +
!
 +
 
 +
|-
 +
| *primer mix: 1 ?L forward primer+ 1 ?L reverse primer +18 ?L MQH2O. Primers varied for each gRNA
 +
 
 +
|-
 +
| *control consisted of DNA only, no primers
 +
 
 +
|-
 +
| Thermocycler conditions:
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
Initial Denature 98 95°C/30 sec
Initial Denature 98 95°C/30 sec
Denature 98 95°C/30 sec
Denature 98 95°C/30 sec
Line 1,119: Line 2,687:
# of cycles 15 cycles
# of cycles 15 cycles
Final Extension 72 3 min
Final Extension 72 3 min
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| 7/11/2014
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| All PCR gRNAs DPN1 digested:
 +
 
 +
|-
 +
| 1. Add 1 ?l of the Dpn I restriction enzyme (10 U/?l) directly to each
 +
 
 +
|-
 +
| amplification reaction below the mineral oil overlay.
 +
 
 +
|-
 +
| 2. Gently and thoroughly mix each reaction mixture by pipetting the
 +
 
 +
|-
 +
| solution up and down several times. Spin down the reaction mixtures in
 +
 
 +
|-
 +
| a microcentrifuge for 1 minute and immediately incubate each reaction
 +
 
 +
|-
 +
| at 37°C for 1 hour to digest the parental (i.e., the nonmutated)
 +
 
 +
|-
 +
| supercoiled dsDNA.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Transformation done to protocol for all gRNAs into DH5?. Full volume (250?l) plated onto Amp  plates and  incubated o/n.
 +
 
 +
|-
 +
| Week 10
 +
 
 +
|-
 +
| 7/16
 +
 
 +
|-
 +
| O/N cultures of SDM gRNA p51 (4 colonies) and SC1 (2 colonies)
 +
 
 +
|-
 +
| 7/17
-
7/11/2014
+
|-
 +
| Mini prepped all p51 and SC1 cultures to protocol and then nano dropped:
-
All PCR gRNAs DPN1 digested:
+
|-
-
1. Add 1 μl of the Dpn I restriction enzyme (10 U/μl) directly to each
+
| 
-
amplification reaction below the mineral oil overlay.
+
-
2. Gently and thoroughly mix each reaction mixture by pipetting the
+
-
solution up and down several times. Spin down the reaction mixtures in
+
-
a microcentrifuge for 1 minute and immediately incubate each reaction
+
-
at 37°C for 1 hour to digest the parental (i.e., the nonmutated)
+
-
supercoiled dsDNA.
+
-
Transformation done to protocol for all gRNAs into DH5α. Full volume (250μl) plated onto Amp  plates and  incubated o/n.
+
|-
-
Week 10
+
|
-
7/16
+
-
O/N cultures of SDM gRNA p51 (4 colonies) and SC1 (2 colonies)
+
-
7/17
+
-
Mini prepped all p51 and SC1 cultures to protocol and then nano dropped:
+
-
 
+
-
_t Sample ng/ μL 260/280 260/230
+
|-
 +
|}
p51 (1) 62.5 1.90 1.65
p51 (1) 62.5 1.90 1.65
     p51(2) 87.2 1.95 2.20
     p51(2) 87.2 1.95 2.20
Line 1,147: Line 2,759:
sc1 (1) 126.5 1.83 1.48
sc1 (1) 126.5 1.83 1.48
sc1(2) 101.9 1.88 1.84
sc1(2) 101.9 1.88 1.84
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
 +
|-
 +
|
-
Restriction digest done to protocol:
+
|-
 +
|
-
_t Sample Buffer Green Fast Digest Enzyme Mix* DNA
+
|-
 +
| Restriction digest done to protocol:
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
(200 ng) MQ H2O Total
(200 ng) MQ H2O Total
Volume
Volume
-
p51 (1) 1.0 μL 1.0 μL 3.0 μL 5.0 μL 10.0 μL
+
p51 (1) 1.0 ?L 1.0 ?L 3.0 ?L 5.0 ?L 10.0 ?L
-
p51 (2) 1.0 μL 1.0 μL 2.0 μL 6.0 μL 10.0 μL
+
p51 (2) 1.0 ?L 1.0 ?L 2.0 ?L 6.0 ?L 10.0 ?L
-
p51 (3) 1.0 μL 1.0 μL 2.0 μL 6.0 μL 10.0 μL
+
p51 (3) 1.0 ?L 1.0 ?L 2.0 ?L 6.0 ?L 10.0 ?L
-
p51 (4) 1.0 μL 1.0 μL 3.0 μL 5.0 μL 10.0 μL
+
p51 (4) 1.0 ?L 1.0 ?L 3.0 ?L 5.0 ?L 10.0 ?L
-
SC1 (1) 1.0 μL 1.0 μL 2.0 μL 6.0 μL 10.0 μL
+
SC1 (1) 1.0 ?L 1.0 ?L 2.0 ?L 6.0 ?L 10.0 ?L
-
SC1 (2) 1.0 μL 1.0 μL 2.0 μL 6.0 μL 10.0 μL
+
SC1 (2) 1.0 ?L 1.0 ?L 2.0 ?L 6.0 ?L 10.0 ?L
-
_t
+
{| class = "wikitable"
-
*Enzyme Mix contains 1.0 μL Spe1 + 1.0 μL BgI1 + 23.0 μL mqH2O
+
|-
-
Samples evaporated after digesting to a total volume of around 4 μL
+
!
-
Run on a 1% Gel. The following lists the sample loaded into each lane.
+
|-
-
_t Lane Sample
+
| *Enzyme Mix contains 1.0 ?L Spe1 + 1.0 ?L BgI1 + 23.0 ?L mqH2O
 +
 
 +
|-
 +
| Samples evaporated after digesting to a total volume of around 4 ?L
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Run on a 1% Gel. The following lists the sample loaded into each lane.
 +
 
 +
|-
 +
|}
1 2-Log DNA Ladder
1 2-Log DNA Ladder
2 SDM p51 (1)
2 SDM p51 (1)
Line 1,174: Line 2,810:
6 Blank
6 Blank
7 SDM P51 (4)
7 SDM P51 (4)
-
_t
+
{| class = "wikitable"
-
[[File:UCB-induced cc9-gel-140717.JPG]]
+
|-
 +
!
-
7/19/2014
+
|-
-
PCR amplify p50
+
| [[File:UCB-induced cc9-gel-140717.JPG]]
-
_t Reactants Volume
+
|-
-
Q5 Master Mix 12.5 μL
+
|
-
10 μM forward primer 1.5 μL
+
-
10 μM reverse primer 1.5 μL
+
-
DNA p50 (1:50) 1.0 μL
+
-
Nuclease Free H2O 9.0 μL
+
-
Final Reaction Volume 25.0 μL
+
-
_t
+
-
7/21/2014
+
-
Restriction digest same gRNAs again
+
|-
 +
| 7/19/2014
 +
|-
 +
| PCR amplify p50
-
_t Sample Buffer cutsmart Enzyme Xbal Enzyme Spe1 DNA
+
|-
 +
|
 +
 
 +
|-
 +
|}
 +
Q5 Master Mix 12.5 ?L
 +
10 ?M forward primer 1.5 ?L
 +
10 ?M reverse primer 1.5 ?L
 +
DNA p50 (1:50) 1.0 ?L
 +
Nuclease Free H2O 9.0 ?L
 +
Final Reaction Volume 25.0 ?L
 +
{| class = "wikitable"
 +
|-
 +
!
 +
 
 +
|-
 +
| 7/21/2014
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Restriction digest same gRNAs again
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
|}
(200 ng) MQ H2O Total
(200 ng) MQ H2O Total
Volume
Volume
-
p51 (1) 2.0 μL 1.0 μL 1.0 μL 3.2 μL 12.8 μL 20.0 μL
+
p51 (1) 2.0 ?L 1.0 ?L 1.0 ?L 3.2 ?L 12.8 ?L 20.0 ?L
-
p51 (2) 2.0 μL 1.0 μL 1.0 μL             2.3 μL 13.7 μL 20.0 μL
+
p51 (2) 2.0 ?L 1.0 ?L 1.0 ?L             2.3 ?L 13.7 ?L 20.0 ?L
-
p51 (3) 2.0 μL 1.0 μL 1.0 μL 1.8 μL 14.2 μL 20.0 μL
+
p51 (3) 2.0 ?L 1.0 ?L 1.0 ?L 1.8 ?L 14.2 ?L 20.0 ?L
-
p51 (4) 2.0 μL 1.0 μL 1.0 μL 3.0 μL 13.0μL 20.0 μL
+
p51 (4) 2.0 ?L 1.0 ?L 1.0 ?L 3.0 ?L 13.0?L 20.0 ?L
-
SC1 (1) 2.0 μL 1.0 μL 1.0 μL 1.6 μL 14.4 μL 20.0 μL
+
SC1 (1) 2.0 ?L 1.0 ?L 1.0 ?L 1.6 ?L 14.4 ?L 20.0 ?L
-
SC1 (2) 2.0 μL 1.0 μL 1.0 μL 2.0 μL 14.0μL 20.0 μL
+
SC1 (2) 2.0 ?L 1.0 ?L 1.0 ?L 2.0 ?L 14.0?L 20.0 ?L
-
P51 2.0 μL 1.0 μL 1.0 μL 4.0 μL 12.0μL
+
P51 2.0 ?L 1.0 ?L 1.0 ?L 4.0 ?L 12.0?L
-
P51 2.0 μL 1.0 μL 1.0 μL 4.0 μL 12.0μL
+
P51 2.0 ?L 1.0 ?L 1.0 ?L 4.0 ?L 12.0?L
-
p51 2.0 μL 1.0 μL 1.0 μL 4.0 μL 12.0μL
+
p51 2.0 ?L 1.0 ?L 1.0 ?L 4.0 ?L 12.0?L
-
_t
+
{| class = "wikitable"
 +
|-
 +
!
-
Enzymes are used as a 1:25 dilution
+
|-
 +
|
-
Run on a 1% Gel. The results were still unclear, the following day samples were sent for sequencing.
+
|-
 +
| Enzymes are used as a 1:25 dilution
-
Week 11
+
|-
 +
|
-
7/23/2014
+
|-
-
Sent p51 SDM 1, 3, 4 and p51 original as a control for sequencing. Confirmation that SDM was successful and p51 no longer has the restriction site spe1 in the gRNA location.  
+
| Run on a 1% Gel. The results were still unclear, the following day samples were sent for sequencing.  
-
Sequencing: Total volume 0f 15 μL
+
|-
-
_t SDM Product DNA μL Primer p51seq5 MQ H2O
+
|
-
1 SDM1p51 8.0μL 2.5 μL 4.5μL
+
-
2 SDM3p51 4.4μL 2.5μL 8.1μL
+
-
3 SDM4p51 7.5μL 2.5μL 5.0μL
+
-
4 p51 control 4.2μL 2.5μL 8.3μL
+
-
_t
+
-
Mini prepped 10 o/n cultures of SDM of p50. Done to protocol. Glycerol stocks make of all 10 cultures.  Mini prepped previous gibson colonies 1 and 2.  SDM of p51 for all samples were confirmed
+
|-
 +
| Week 11
-
  Nanodrops were taken to assess the concentration and purity of the samples:
+
|-
-
_t Sample ng/ μL 260/280 260/230
+
|
 +
 
 +
|-
 +
| 7/23/2014
 +
 
 +
|-
 +
| Sent p51 SDM 1, 3, 4 and p51 original as a control for sequencing. Confirmation that SDM was successful and p51 no longer has the restriction site spe1 in the gRNA location.
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Sequencing: Total volume 0f 15 ?L
 +
 
 +
|-
 +
|}
 +
1 SDM1p51 8.0?L 2.5 ?L 4.5?L
 +
2 SDM3p51 4.4?L 2.5?L 8.1?L
 +
3 SDM4p51 7.5?L 2.5?L 5.0?L
 +
4 p51 control 4.2?L 2.5?L 8.3?L
 +
{| class = "wikitable"
 +
|-
 +
!
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Mini prepped 10 o/n cultures of SDM of p50. Done to protocol. Glycerol stocks make of all 10 cultures.  Mini prepped previous gibson colonies 1 and 2.  SDM of p51 for all samples were confirmed
 +
 
 +
|-
 +
|
 +
 
 +
|-
 +
| Nanodrops were taken to assess the concentration and purity of the samples:
 +
 
 +
|-
 +
|}
p50 123.7 1.84 2.15
p50 123.7 1.84 2.15
     SDM1 179.6 1.88 2.19
     SDM1 179.6 1.88 2.19
Line 1,245: Line 2,947:
Restriction digest done to protocol:  
Restriction digest done to protocol:  
-
_t Sample Buffer Cutsmart Enzyme Mix* DNA (200ng) MQ H2O Total Volume
+
{| class = "wikitable"
-
SDM p50 (1) 2.0 μL 1.0 μL 1.2 μL 15.8 μL 20.0 μL
+
|-
-
SDM p50 (2) 2.0 μL 1.0 μL 2.0 μL 15.0 μL 20.0 μL
+
!  Sample
-
SDM p50 (3) 2.0 μL 1.0 μL 1.4 μL 15.6 μL 20.0 μL
+
! Buffer Cutsmart
-
SDM p50  (4) 2.0 μL 1.0 μL 1.2 μL 15.8μL 20.0 μL
+
! Enzyme Mix*
-
SDM p50  (5) 2.0 μL 1.0 μL 2.0 μL 15.0 μL 20.0 μL
+
! DNA (200ng)
-
SDM p50 (6) 2.0 μL 1.0 μL 1.2 μL 15.8 μL 20.0 μL
+
! MQ H2O
-
SDM p50 (7) 2.0 μL 1.0 μL 1.2 μL 15.8μL 20.0 μL
+
! Total Volume
-
SDM p50 (8) 2.0 μL 1.0 μL 1.0 μL 16.0μL 20.0 μL
+
 
-
SDM p50 (9) 2.0 μL 1.0 μL 1.0 μL 16.0μL 20.0 μL
+
|-
-
SDM p50 (10) 2.0 μL 1.0 μL 1.5 μL 15.5μL 20.0 μL
+
| SDM p50 (1)
-
p50control 2.0 μL 1.0 μL 2.0 μL 15.0μL 20.0 μL
+
| 2.0 ?L
-
_t
+
| 1.0 ?L
-
*Enzyme Mix contains 1.0 μL Spe1 + 1.0 μL Xba1 + 48.0 μL mqH2O
+
| 1.2 ?L
 +
| 15.8 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50 (2)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 15.0 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50 (3)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.4 ?L
 +
| 15.6 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50  (4)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.2 ?L
 +
| 15.8?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50  (5)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 15.0 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50 (6)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.2 ?L
 +
| 15.8 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50 (7)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.2 ?L
 +
| 15.8?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50 (8)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.0 ?L
 +
| 16.0?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50 (9)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.0 ?L
 +
| 16.0?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| SDM p50 (10)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.5 ?L
 +
| 15.5?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| p50control
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 15.0?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
|}
 +
*Enzyme Mix contains 1.0 ?L Spe1 + 1.0 ?L Xba1 + 48.0 ?L mqH2O
Restriction Digest for gibson done to protocol:
Restriction Digest for gibson done to protocol:
-
_t Sample Buffer D Enzyme EcoRV DNA
+
{| class = "wikitable"
-
(200 ng) MQ H2O Total
+
|-
-
Volume
+
!  Sample
-
Gibson 1 2.0 μL 1.0 μL 2.0 μL 15.0 μL 20.0 μL
+
! Buffer D
-
Gibson 2 2.0 μL 1.0 μL 1.2 μL 15.8 μL 20.0 μL
+
! Enzyme EcoRV
-
_t
+
! DNA
 +
 
 +
|-
 +
| (200 ng)
 +
| MQ H2O
 +
| Total
 +
 
 +
|-
 +
| Volume
 +
 
 +
|-
 +
| Gibson 1
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 15.0 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
| Gibson 2
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.2 ?L
 +
| 15.8 ?L
 +
| 20.0 ?L
 +
 
 +
|-
 +
|}
*EcoRv dilution (1:25)
*EcoRv dilution (1:25)
Run on a 1% Gel. The following lists the sample loaded into each lane.
Run on a 1% Gel. The following lists the sample loaded into each lane.
-
_t Lane Sample
+
{| class = "wikitable"
-
1 2-Log DNA Ladder
+
|-
-
2 SDM p50 (1)
+
!  Lane
-
3 SDM p50 (2)
+
! Sample
-
4 SDM p50 (3)
+
 
-
5 SDM p50 (4)
+
|-
-
6 SDM p50  (5)
+
| 1
-
7 SDM p50 (6)
+
| 2-Log DNA Ladder
-
8 SDM p50 (7)
+
 
-
9 SDM p50 (8)
+
|-
-
10 SDM p50 (9)
+
| 2
-
11 SDM p50 (10)
+
| SDM p50 (1)
-
12 p50 control
+
 
-
13 p50 undigested
+
|-
-
14 Gibson 1
+
| 3
-
15 Gibson 2
+
| SDM p50 (2)
-
16 sb1c3
+
 
-
17 blank
+
|-
 +
| 4
 +
| SDM p50 (3)
 +
 
 +
|-
 +
| 5
 +
| SDM p50 (4)
 +
 
 +
|-
 +
| 6
 +
| SDM p50  (5)
 +
 
 +
|-
 +
| 7
 +
| SDM p50 (6)
 +
 
 +
|-
 +
| 8
 +
| SDM p50 (7)
 +
 
 +
|-
 +
| 9
 +
| SDM p50 (8)
 +
 
 +
|-
 +
| 10
 +
| SDM p50 (9)
 +
 
 +
|-
 +
| 11
 +
| SDM p50 (10)
 +
 
 +
|-
 +
| 12
 +
| p50 control
 +
 
 +
|-
 +
| 13
 +
| p50 undigested
 +
 
 +
|-
 +
| 14
 +
| Gibson 1
 +
 
 +
|-
 +
| 15
 +
| Gibson 2
 +
 
 +
|-
 +
| 16
 +
| sb1c3
 +
 
 +
|-
 +
| 17
 +
| blank
 +
 
 +
|-
 +
|
-
_t
+
|-
 +
|}
[[File:UCB-induced cc9-gel-140723.JPG]]
[[File:UCB-induced cc9-gel-140723.JPG]]
Line 1,298: Line 3,171:
Restriction digest done to protocol:  
Restriction digest done to protocol:  
-
_t Sample Buffer Cutsmart Enzyme Mix* DNA
+
{| class = "wikitable"
-
(250 ng) MQ H2O Total
+
|-
-
Volume
+
!  Sample
-
SDM p50 (1) 2.0 μL 1.0 μL 1.5 μL 20.0 μL 25.0 μL
+
! Buffer Cutsmart
-
SDM p50 (2) 2.0 μL 1.0 μL 2.0 μL 19.5 μL 25.0 μL
+
! Enzyme Mix*
-
SDM p50 (3) 2.0 μL 1.0 μL 1.8 μL 19.7 μL 25.0 μL
+
! DNA
-
SDM p50  (4) 2.0 μL 1.0 μL 1.5 μL 20.0μL 25.0 μL
+
 
-
SDM p50  (5) 2.0 μL 1.0 μL 2.0 μL 19.5 μL 25.0 μL
+
|-
-
SDM p50 (6) 2.0 μL 1.0 μL 1.5 μL 20.0 μL 25.0 μL
+
| (250 ng)
-
SDM p50 (10) 2.0 μL 1.0 μL 2.0 μL 19.5μL 25.0 μL
+
| MQ H2O
-
p50control 2.0 μL 1.0 μL 2.0 μL 19.5μL 25.0 μL
+
| Total
-
_t
+
 
-
*Enzyme mix: 5.5μL Xba1 + 5.5μL Spe1 for a total volume of 11μL
+
|-
 +
| Volume
 +
 
 +
|-
 +
| SDM p50 (1)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.5 ?L
 +
| 20.0 ?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
| SDM p50 (2)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 19.5 ?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
| SDM p50 (3)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.8 ?L
 +
| 19.7 ?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
| SDM p50  (4)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.5 ?L
 +
| 20.0?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
| SDM p50  (5)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 19.5 ?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
| SDM p50 (6)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 1.5 ?L
 +
| 20.0 ?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
| SDM p50 (10)
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 19.5?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
| p50control
 +
| 2.0 ?L
 +
| 1.0 ?L
 +
| 2.0 ?L
 +
| 19.5?L
 +
| 25.0 ?L
 +
 
 +
|-
 +
|}
 +
*Enzyme mix: 5.5?L Xba1 + 5.5?L Spe1 for a total volume of 11?L
Run on a 1% Gel. Results were not as anticipated. Restriction Ligation was repeated.
Run on a 1% Gel. Results were not as anticipated. Restriction Ligation was repeated.
Line 1,316: Line 3,258:
Restriction Ligation
Restriction Ligation
-
_t Sample Buffer Cutsmart EcoR1 Xba1 Spe1 Pst1 DNA
+
{| class = "wikitable"
-
(200 ng) MQ H2O Total
+
|-
-
Volume
+
!  Sample
-
SDM p50 (1) 5.0 μL -- 1.0 μL -- 1.0 μL 1.0 μL 42.0 μL 50.0 μL
+
! Buffer Cutsmart
-
SDM p50 (4) 5.0 μL -- 1.0 μL -- 1.0 μL       1.0 μL 42.0 μL 50.0 μL
+
! EcoR1
-
SDM p50 (6) 5.0 μL -- 1.0 μL -- 1.0 μL 1.0 μL 42.0 μL 50.0 μL
+
! Xba1
-
Litimus28i 5.0 μL -- -- 1.0 μL 1.0 μL 1.5 μL 41.5μL 50.0 μL
+
! Spe1
-
Litimus+Ic3 5.0 μL 1.0 μL -- -- 1.0 μL 0.8 μL 42.2 μL 50.0 μL
+
! Pst1
-
PsBGA1 (RFP) 5.0 μL 1.0 μL -- 1.0 μL -- 3.8 μL 39.2 μL 50.0 μL
+
! DNA
-
_t
+
 
 +
|-
 +
| (200 ng)
 +
| MQ H2O
 +
| Total
 +
 
 +
|-
 +
| Volume
 +
 
 +
|-
 +
| SDM p50 (1)
 +
| 5.0 ?L
 +
| --
 +
| 1.0 ?L
 +
| --
 +
| 1.0 ?L
 +
| 1.0 ?L
 +
| 42.0 ?L
 +
| 50.0 ?L
 +
 
 +
|-
 +
| SDM p50 (4)
 +
| 5.0 ?L
 +
| --
 +
| 1.0 ?L
 +
| --
 +
| 1.0 ?L
 +
|       1.0 ?L
 +
| 42.0 ?L
 +
| 50.0 ?L
 +
 
 +
|-
 +
| SDM p50 (6)
 +
| 5.0 ?L
 +
| --
 +
| 1.0 ?L
 +
| --
 +
| 1.0 ?L
 +
| 1.0 ?L
 +
| 42.0 ?L
 +
| 50.0 ?L
 +
 
 +
|-
 +
| Litimus28i
 +
| 5.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 1.0 ?L
 +
| 1.5 ?L
 +
| 41.5?L
 +
| 50.0 ?L
 +
 
 +
|-
 +
| Litimus+Ic3
 +
| 5.0 ?L
 +
| 1.0 ?L
 +
| --
 +
| --
 +
| 1.0 ?L
 +
| 0.8 ?L
 +
| 42.2 ?L
 +
| 50.0 ?L
 +
 
 +
|-
 +
| PsBGA1 (RFP)
 +
| 5.0 ?L
 +
| 1.0 ?L
 +
| --
 +
| 1.0 ?L
 +
| --
 +
| 3.8 ?L
 +
| 39.2 ?L
 +
| 50.0 ?L
 +
 
 +
|-
 +
|}
Sequenced SDMp50 was not the correct product, the above project did not continue.
Sequenced SDMp50 was not the correct product, the above project did not continue.
Line 1,336: Line 3,354:
O/D recordings:  
O/D recordings:  
-
_t Time MG Bw+kan Bw(-)kan
+
{| class = "wikitable"
-
11:00 .042 .026 .071
+
|-
-
11:34 .086 .035 .088
+
!  Time
-
12:10 .239 .078 .223
+
! MG
-
12:20 .328 .098 .296
+
! Bw+kan
-
12:28 -- -- .325
+
! Bw(-)kan
-
12:47 -- .196 --
+
 
-
1:10 -- .308 --
+
|-
-
1:15 -- .320 --
+
| 11:00
-
_t
+
| .042
 +
| .026
 +
| .071
 +
 
 +
|-
 +
| 11:34
 +
| .086
 +
| .035
 +
| .088
 +
 
 +
|-
 +
| 12:10
 +
| .239
 +
| .078
 +
| .223
 +
 
 +
|-
 +
| 12:20
 +
| .328
 +
| .098
 +
| .296
 +
 
 +
|-
 +
| 12:28
 +
| --
 +
| --
 +
| .325
 +
 
 +
|-
 +
| 12:47
 +
| --
 +
| .196
 +
| --
 +
 
 +
|-
 +
| 1:10
 +
| --
 +
| .308
 +
| --
 +
 
 +
|-
 +
| 1:15
 +
| --
 +
| .320
 +
| --
 +
 
 +
|-
 +
|}
The cells were all pulled at the time they were at an O/D  nearest .325. All O/D’s were diluted to .30
The cells were all pulled at the time they were at an O/D  nearest .325. All O/D’s were diluted to .30
Once the cells were made competent they were transformed using a gRNA  target, gRNA scramble, and  a sb1c3  strain.
Once the cells were made competent they were transformed using a gRNA  target, gRNA scramble, and  a sb1c3  strain.

Revision as of 23:55, 13 October 2014

Week 1 5/12/14 

Made overnight cultures of….

- Qi gRNA P44251 (AmpR) NOTE: plate from Andrew is labeled P44231 - Qi wr cas9 P44250 (AmpR) - BW 23115 Kan casA (KanR)

5/13/14 Glycerol stocks - Stocks made from overnight cultures (3 from each) - 500 ?L bacteria + 500 ?L 40% glycerol stock

  • Glass tube containing #P44250 broke and was discarded – overnight repeated

Mini Prep Protocol done per manufacturer’s instructions with following exceptions: -Done with P44250 on 5/13/14 -Done with P44251 on 5/14/14 -Step 1: Cultures split into 3 separate 1.5 mL aliquots. Supernatant was removed from all three, pellets resuspended in a small amount of EB Buffer and moved into single tube. Centrifuged again and supernatant was removed before proceeding to Step 2.

5/14/14

Nano Drop of Mini Prep
Sample 260/280 Ratio 260/230 Ratio DNA Concentration
P44250 1.94 2.17 49.4 ng/?L
P44251 1.94 2.47 52.7 ng/?L
  • Samples were blanked with EB Buffer

Restriction Digest - NEBcutter V2.0 used to check digestion cut sites (done in silico) - Digest reaction performed as shown below (from NEB) - Incubated for 1 hour at 37°C - No heat inactivation performed


P44250 P44251
Cutsmart Buffer 5.0 ?L 5.0 ?L
XbaI 1.0 ?L 1.0 ?L
XhoI 1.0 ?L --
SpeI 1.0 ?L --
EcoRI-HF -- 1.0 ?L
DNA (200 ng) 4.0 ?L 4.0 ?L
MQ H2O 38.0 ?L 39.0 ?L
Total Volume 50.0 ?L 50.0 ?L

Agarose Gel Electrophoresis -1% agarose gel prepared -2 ?L loading dye added to 18 ?L of each sample for loading into each lane -1Kb+ DNA Ladder used -Overnight staining in EtBr done to better visualize faint bands

Lane 1: 2-Log DNA Ladder Lane 2: P44250 digest Lane 3: P44251 digest

P50 plasmid should have 3 segments after digestion: 4221 bp, 1740 bp, 970 bp P51 plasmid should have 2 segments after digestion: 540 bp, 2045 bp -This is demonstrated in the gel image shown below. UCB-incuced cc9 gel-140514-.jpg


Plate Streaking -Used to check the TetR gene in the P44250 plasmid -2 mL LB containing 50 mg/ ?L Tet added to plate and allowed to soak in -Plates left at room temperature overnight

- LB only plate – negative control - Amp plate - Amp + Tet - Tet


5/15/14 Dry Lab - gRNA designed for Kanamycin - Plasmid design – considering Gibson assembly and cPEC

Analysis of streak plates from 5/14/15 Results: (See Below) LB: Slightly less growth than Amp plate Amp plate: Normal Growth Amp + Tet: No bacteria present Tet: No bacteria present Results were inconsistent with expectations. There is some concern regarding the potency of the tetracycline used. Further research revealed that the TetR gene is a repressor for TetO which part of the bi-directional promoter for cas9. Thus TetR is inhibited with presence of tetracycline. We will use TetR as a regulator for cas9.

UCB-induced cc9- plates-140515.jpg

5/16/14 Dry Lab - gRNA designs modified - Primer design for Gibson assembly of our plasmid

Week 2 5/19/14 Transformation: 1 ?L P44250 (cas9) plasmid added to K12 E. coli strain Heat shock done according to protocol (45 sec @ 42°C) Cultures incubated at 37°C for 1.5 hours 4 Plates with K12 and the plasmid were plated by streaking as follows: - Amp, Amp (? plate with 1:10 dilution, ? plate 1:1), Amp + Kan, Amp + Kan (? plate with 1:10 dilution, ? plate 1:1) 5/20/14 Plates from 5/19/14 (shown below).

UCB-induced cc9-plates-140520.jpg


Two individual colonies were taken and grown overnight from the Amp plate (marked with “x” in upper left plate of above image). There was no growth on the two plates with Amp and Kan (K12 strain does not contain Kan resistance).

5/22/14 Transformation: (Chemically competent BWF+ cells with P44250 plasmid) 1 ?L P44250 (cas9) plasmid added to conjugated BWF+ strain Transformation protocol followed with following exceptions: -200 ?L SOC added to each sample -Incubated for 2 hours -1:10 dilution made (10 ?L into 90 ?L SOC) -Cultures plated on Amp plates, one with full concentration and another with the 1:10 dilution


5/23/14 Arrival of primers previously ordered All were reconstituted in MQ H2O to 100 ?M concentration

Results of plates from 5/22/14: - BFW+ w/ P50 plasmid: growth at full and 1:10 dilution (Top left of image) - BFW+ w/ P50 plasmid full strength: No growth (Bottom left of image) - BW only: full strength showed minor growth (Bottom right of image) - BW only: No growth at full and 1:10 dilution (Top right of image) (*This was an unexpected result – it may be that these two plates were mislabeled) UCB-Induced cc9-plates-140523.jpg


Single colonies taken from the BFW+ w/ P50 plasmid plate in the 1:10 dilution and placed in 2 ?L liquid culture overnight with the following antibiotics: - AMP - Kan - AMP + Kan - Tet


5/24/14 As shown the image below, overnight cultures from 5/23/14 are all cloudy demonstrating bacterial growth. UCB-induced cc9- culture-140524.jpg


PCR Reaction – Used to amplify out cas9 from P44250 plasmid and CMR from PSB1C3 plasmid *DNA samples diluted so each contains 0.5 ng/?L

Cas9 CMR
Q5 Master Mix volume 25 ?L 25 ?L
dcas9 Primer volume 2.5 ?L --
dCMR Primer volume -- 2.5 ?L
DNA added 2.5 ?L 2.5 ?L
Q5 Polymerase volume 0.5 ?L 0.5 ?L
Nuclease Free H2O 20 ?L 20 ?L
Final Reaction Volume 50.0 ?L 50.0 ?L

PCR Settings: 30 cycles as follows - 98 °C Denature 30 sec - 70 °C Anneal 30 sec - 72 °C Extension 2.5 min

Week 3

5/25/14 Gel electrophoresis performed on PCR reactions from 5/24/14. 1% gel made from 0.514 g agarose with 50 mL 1X TAE and 5 ?L EtBr. Lane 1: 5 ?L 2-Log DNA Ladder Lane 2: CMR sample* (~2 Kb size) Lane 3: Cas9 sample* (~5 Kb size)

  • Each contained 8 ?L reaction sample + 2 ?L loading dye

Gel Results:

Lane 1 – Ladder Lane 2 – Although band of correct size visible for CMR sample (~2Kb), there may be contamination Lane 3 – No bands visible – This is perhaps due to having the extension time too short in the PCR reaction (Advisors suggest 60 sec per 1000 bases).

UCB-incuced cc9-gel-140525.jpg


5/27/14 Repeat of PCR using same protocol as listed on 5/24/14 except for different times on the thermocycler as follows:

Cas9 CMR
Initial Denature 98°C/2 min 98°C/2 min
Denature 98°C/30 sec 98°C/30 sec
Anneal 69°C/30 sec 72°C/30 sec
Extend 69°C/ 2.5 min 72°C/2.5 min
Final Extension 72°C/6 min 72°C/5 min
  • Thermocycler containing CMR stopped after 1 cycle and held at 98°C for ~3hours. Machine was reset and run. This sample may no longer be viable.


5/28/14 Dry lab – primer designs Repeat of PCR for the CMR reaction, same as described on 5/24/14 and shown below.

Reactants CMR
Q5 Master Mix 25 ?L
dCMR Primer 2.5 ?L
Q5 Polymerase 0.5 ?L
CMR DNA 2.0 ?L
Nuclease Free H2O 20 ?L
Final Reaction Volume 50.0 ?L

5/29/14 Gel electrophoresis: 1% gel ran at 110V for ~40 minutes.

Lane Sample Amount Loading dye amount
1 2-Log DNA Ladder 0.5  ?L --
2 Cas9 5.0  ?L 1.0  ?L
3 CMR 1 (from 5/24) 5.0  ?L 1.0  ?L
4 CMR 2 (from 5/27) 5.0  ?L 1.0  ?L
5 CMR 3 (from 5/28) 5.0  ?L 1.0  ?L

Results from Gel (shown below): Lane 2: Cas9 band visible of appropriate size (approximately 5,000 bp) Lane 3: CMR 1- slight smearing Lane 4: CMR 2 – smearing present Lane 5: CMR 3- clean, no smearing All CMR lanes have band present of correct size (approx. 2,000 bp)

UCB-induced cc9-gel-140529.jpg

Gel extraction performed on cas9, CMR1, and CMR2 lanes using E.Z.N.A. Gel extraction kit following the protocol. Following amounts: - Cas9: 110 ?L buffer with 0.1103 g sample - CMR1: 53 ?L buffer with 0.0533 g sample - CMR2: 90 ?L buffer with 0.0904 g sample

Nanodrops performed to determine DNA concentration and purity with the following results:


Sample ng/ ?L 260/280 260/230 **ng/ ?L **260/280 **260/230
Gel-Cas9 7.3 2.20 0.57 5.2 2.06 0.52
Cas9 483.2 1.78 0.72 36.3 1.85 1.72
Gel-CMR1 9.6 2.02 0.63 6.5 2.35 0.90
Gel-CMR2 8.3 2.06 0.83 5.0 2.21 0.69
CMR2 618.8 1.66 0.61 44.5 1.89 1.41
CMR 3 535.8 1.73 0.61 17.8 1.89 1.31
CMR1 516.6 1.69 0.61 68.5 1.84 1.74
  • Trial 2 completed on 5/30/14 shown in these columns

Values for the ratios should be between 2.0 and 2.2 for a pure sample. The values for Trial #1 are much too low. In order to increase sample purity, a DNA cleanup is performed following the protocol given in the E.Z.N.A. gel extraction kit in amounts shown below. - 25 ?L samples - 25 ?L binding buffer - Washed 2x with 700 ?L SPW - 30 ?L elution buffer added, allowed to rest 4 minutes - Centrifuged on max for 2 min


5/30/14 After the DNA cleanup protocol – nanodrop performed again with results listed in above table as Trial #2

Restriction Digestion performed on Cas9 and CMR1 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts: 26 ?L sample 1.0 ?L DPN1 (100 fold dilution) 3.0 ?L 10X CutSmart Buffer

Gibson Assembly performed to join pSB1C3 backbone with cas9 PCR product. Protocol for Gibson was followed for the reaction.

Reactants Volume
Gibson Master Mix 10 ?L
Cas9 1.6 ?L (50 ng)
Cmr 2.4 ?L (143 ng)
Nuclease Free H2O 6 ?L
Final Reaction Volume 20.0 ?L

Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.

Week 4

6/2/14

Grow Gibson colonies in 6 mL LB media + 30 ?L chloroamp

Transform Gibson colonies following protocol and with amounts as shown: - BWF+ w/cas9: 15 ?L into 135 ?L SOC - BW w/cas9: 15 ?L into 135 ?L SOC - BWF+ (no cas9): 30 ?L into 270 ?L SOC - BW (no cas9): 30 ?L into 270 ?L SOC

90 ?L of the above four reactions plated as follows and incubated: - BWF+ w/cas9: Amp + Kan - BW w/cas9: Amp + Kan - BWF+ (no cas9): (2 Plates) Amp + Kan and Kan only - BW (no cas9): (2 Plates) Amp + Kan and Kan only - All four reactions also plated together on Quad plate with Chloramp - BWF+ (Not transformed) on Kan + Tet (plated both at full strength and as 1:10 dilution)

6/3/14

Analysis of Plates from 6/2/14: Only one colony was present on Gibson plate (BWF+ w/cas9) This colony was taken and suspended in O/N culture (LB and Choroamp) MiniPrep performed on the O/N cultures *Renamed the product GAcasC (Gibson Assembly w/cas and Chloroamp) NanoPrep on GAcasC

ng/ ?L 260/280 260/230
201.0 1.88 2.24

Digest performed according to protocol on GAcasC and P50 plasmid using EcoRV and BamHI as follows:

Reactants GAcasC P50
Buffer 3 2.5 ?L 2.5 ?L
EcoRV 0.5 ?L 0.5 ?L
BamHI 0.5 ?L 0.5 ?L
BSA (1:4 dilution) 1.0 ?L 1.0 ?L
DNA (250 ng) 1.25 ?L 5.0 ?L
MQ H2O 19.25 ?L 15.5 ?L
Total Volume 25.0 ?L 25.0 ?L

1% Gel made for electrophoresis. Each sample had 1 ?L of loading dye added, 5 ?L of DNA ladder and 5 ?L of each sample was loaded. Gel ran 120V for ~1 hour.

Well Loaded
1 DNA Ladder
2 GAcasC digest
3 CmR1 Dpn1 product
4 Cas9 Dpn1 product
5 P50 digestion product

Gel Results: Lane 2 should have multiple products which are not present in the gel. The other lanes are as expected.

UCB-induced cc9-gel-140603.jpg


PCR using Q5 is used to amplify the P51 CRISPR backbone using primers CRE1 & CRE4 provided by Andrew. Primers were diluted 1:10 from 100 mM stock

Reactants Volume
Q5 Master Mix 25 ?L
CRE1 Primer 2.5 ?L
CRE4 Primer 2.5 ?L
P51 DNA 2.0 ?L
Q5 Polymerase 0.5 ?L
Nuclease Free H2O 17.5 ?L
Final Reaction Volume 50.0 ?L

Thermocycler set as follows for the reaction:

Initial Denature 98°C/30 sec
Denature 98°C/20 sec
Anneal 60°C/20 sec
Extend 72°C/3 min

Restriction Digestion performed on P51 with DPN1 (to remove any methylation present) using standard digestion protocol with the following amounts: 26 ?L sample 1.0 ?L DPN1 (100 fold dilution) 3.0 ?L 10X CutSmart Buffer

PCR performed to dimerize each of the gRNA primers previously designed (729, 666, 756, SC1, and SC2) following the cPEC protocol and using the following amounts:

Reactants Volume
Q5 Master Mix 25.0 ?L
Primer 1.7 ?L
Primer 2.0 ?L
Q5 Polymerase 0. 5 ?L
Nuclease Free H2O 19.5 ?L
Final Reaction Volume 50.0 ?L

Nanodrop performed on some of these products (due to timing) to check for purity and concentration (results below). It is assumed that the other samples have high enough DNA levels to proceed.

Sample ng/ ?L 260/280 260/230
729 405.5 1.78 0.67
SC2 393.2 1.80 0.67
CasC 455.2 1.5 0.60

cPEC performed to combine the P51 amplification and the each of the dimerized gRNAs separately following the cPEC protocol and using the following amounts:


Reactants Volume
Q5 Master Mix 12.5 ?L
Insert (our primer dimers) 1.7 ?L
Template DNA 2.0 ?L
Q5 Polymerase 0.25 ?L
Nuclease Free H2O 10.05 ?L
Final Reaction Volume 25.0 ?L

Repeat of Gibson to combine cas9 and CmR (pSB1C3 backbone) with cas9 PCR product. Gibson protocol was followed for the reaction and included a positive control (same as reaction completed on 5/30/14).

Reactants Volume
Gibson Master Mix 10 ?L
Cas9 1.6 ?L (50 ng)
Cmr 2.4 ?L (143 ng)
Nuclease Free H2O 6 ?L
Final Reaction Volume 20.0 ?L

Product was transformed into competent cells following the Gibson transformation protocol that included a positive control.

O/N cell cultures made from plates in order to perform a Tet titration tomorrow. 2 tubes of each were made by taking a single colony and mixing with 5 mL of LB and antibiotics as listed below before incubation.

Sample Antibiotic
BWF+ w/ cas 5 ?L Kan + 10 ?L Amp
BW w/ cas 5 ?L Kan + 10 ?L Amp
BWF+ 5 ?L Kan
BW 5 ?L Kan
BWF+ (not transformed) 5 ?L Kan + 10 ?L Tet

6/4/14

Tet titration on O/N cultures was performed by using different concentrations (from 0-1000 ng/mL) of anhydrotetracycline plus a chloramp control.

  • Cancelled as cell growth had already neared plateau values
Sample 0 ng/mL 0.5 ng/mL 5 ng/mL 20 ng/mL 50 ng/mL 250 ng/mL 1000 ng/mL Chloro
BW 2.38 2.401 2.389 2.382 2.386 2.398 2.373 2.379
BWF+ 2.379 2.376 2.428 2.390 2.378 2.365 2.378 2.386
BWcas 2.391 2.356 2.367 2.355 2.366 2.330 2.362 2.382
BWF+ cas 2.438 2.447 2.448 2.434 2.436 2.415 2.419 2.415
BWF+ Tet 2.135 2.146 2.141 2.152 2.128 2.127 2.134 2.134

New O/N cell cultures made to do the titration on 6/5/14. Same amounts as shown above were used except only one of each was made. No cells added to O/N culture on 6/4/14, that is why procedure was repeated.

6/5/14

Since the Gibson Assembly does not seem to be working, cPEC was performed to combine the CmR backbone with Cas9. Three different cycles were run in an attempt to optimize the protocol; the following amounts were used in all three reactions:


Reactants Volume
Q5 Master Mix 10.0 ?L
Cas9 DNA 5.0 ?L
CmR DNA 5.0 ?L
Nuclease Free H2O 0.00 ?L
Final Reaction Volume 20.0 ?L

Thermocycler was varied for each of the three cPEC reactions as listed below:

cPEC1 cPEC2 cPEC3
Initial Denature 98°C/30 sec 98°C/30 sec 98°C/30 sec
Denature 98°C/10 sec 98°C/20 sec 98°C/10 sec
Anneal 56°C/30 sec 60°C/20 sec 66°C/30 sec
Extend 72°C/90 sec 72°C/30 sec 72°C/90 sec
# of cycles 10 15 10
Final Extension 72°C/10 min 72°C/10 min 72°C/10 min
_t
Nanodrop done on the three cPEC casC reactions with results listed below:

CasC1 489.3 1.80 0.72 CasC2 577.7 1.76 0.59 CasC3 542.5 1.80 0.74 

_t

DNA cleanup done on these three casC cPEC reactions, followed by another nanodrop to examine purity with the results below.


Sample ng/ ?L 260/280 260/230
CasC1 13.4 1.99 1.61
CasC2 11.1 2.02 1.15
CasC3* 9.9 1.84 1.21
_t
1% Gel made for electrophoresis. Each sample had 1 ?L of loading dye added, 5 ?L of DNA ladder and 5 ?L of each sample was loaded. Gel ran 122V for ~50 min.

1 2-Log DNA Ladder 2 cPEC1 3 cPEC2 4 cPEC3 5 GA Control 6 GA Positive Control 7 CasC 8 H2O Control 

_t

UCB-induced cc9- gel-140605.jpg


Titration experiment was done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. It was decided to use BWF+ w/cas9 and w/o cas9 for this experiment. Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.

Time 0 Time 1 Time 2 Time 3 Time 4 Time 5* Time 6
BWF+ 0.041 0.047 0.114 0.283 0.597 0.065 0.171
BWF+ w/cas9 0.035 0.046 0.110 0.262 0.530 0.059 0.154
  • At Time 5, the cultures were again diluted down. The experiment was cancelled after Time 6 due to time constraints and issues with the plates.

6/6/14

Plates with the Gibson Assembly products have shown very slow growth. Seven colonies are visible on the Gibson plate (shown with positive control plate below). UCB-induced cc9-plate-01-140606.jpg UCB-induced cc9-plate-02-140606.jpg

Above left: Gibson Assembly plate with 7 colonies. Above Right: Gibson Assembly positive control plate.

Week 5

6/9/14

Mini-preps were done following manufacturer's protocol except samples were centrifuged 4 minutes at the end (step 9). Ten samples were done that included 7 GA colonies, 1 GA control (labeled GA8), P50, and P51. All were subjected to a nanodrop and a restriction digestion with BamHI and EcoRV was performed on all the samples all except P51. The nanodrop results are shown below:


Sample ng/ ?L 260/280 260/230
GA1 80.5 1.88 2.04
GA2 87.8 1.90 2.14
GA3 53.4 1.89 1.72
GA4 67.1 1.88 2.02
GA5 49.5 1.92 1.86
GA6 84.2 1.90 2.07
GA7 54.6 1.94 2.08
GA8 59.8 1.86 1.60
P50 91.3 1.88 2.12
P51 65.8 1.88 2.03

Samples from the digestion reaction were run on a 1% agarose gel (shown below). From these results, it appears that none of the Gibson Assembly colonies contain the correct products.


Lane Sample
1 2-Log DNA Ladder
2 GA1
3 GA2
4 GA3
5 GA4
6 GA5
7 GA6
8 GA7
9 GA8
10 P50 Digest
11 SB1C3
12 Undigested GA1
13 Undigested GA2
14 Undigested GA3
15 Undigested P50

UCB-induced cc9- gel- 140609.jpg


Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. Working stock of ATC was made and filter sterilized which was used to remake the ATC plates for the titration experiment. 20 plates were made with ? containing Kan and ? containing Amp and Kan. The plates were made with increasing amounts of ATC.

gRNAs from cPEC were transformed and plated according to protocol with controls.


AmilCP restriction digestion was performed on the original and PCR amplified samples and the products run on a 1% agarose gel as shown below:

Reactants PCR AmilCP Original AmilCP
Buffer 3 1.25 ?L 1.25 ?L
BxsI 1.0 ?L 2.5 ?L
DNA (125 ng) 4.0 ?L 1.25 ?L
Nuclease Free H2O 6.25 ?L 7.5 ?L
Final Reaction Volume 12.5 ?L 12.5 ?L

6/10/14

cPEC was performed to join the gRNA (as primer dimers) with the p51 plasmid and AmilCP.

First, primers had to be dimerized. Master mixes of all gRNA primers were created and aliquots made by adding 5 ?L of each to 40 ?L nuclease free H2O (N.F. H2O). Two separate primer mixes were made (one for p51 and one for AmilCP) using 5 ?L of each forward and reverse primer dimer that were added to 40 ?L of N.F. H2O to produce a dilution (1:1:8). 5 ?L of each primer mix was added to 25 ?L of the Q5 master mix and 20 ?L N.F. H2O to make a 50 ?L total reaction for each sample.Products were checked by gel electrophoresis (shown below). UCB-induced cc9- gel-140610.jpg

Next, a gel purification of a PCR P51 product was performed (to select for linear DNA). This product was digested with DpnI. A PCR cleanup was made of other P51 product that was not gel extracted. All of these steps were done following their recommended protocols.

Finally, cPEC reaction was done to combine the gel extracted P51 product with each of the gRNAs using the following amounts. The thermocycler set the same as on 6/3/14:


Reactants Volume
Q5 Master Mix 25.0 ?L
Primer Mix 2.5 ?L
DNA (1:10 dilution) 1.0 ?L
Nuclease Free H2O 25.5 ?L
Final Reaction Volume 54.0 ?L
_t
Following the cPEC reactions, Nanodrops were taken to assess the concentration and purity of the samples:

P51 416.9 1.82 0.67 AmilCP 430.5 1.82 0.67 158 472.2 1.80 0.73 756 479.6 1.78 0.73 666 475.8 1.80 0.73 729 485.4 1.79 0.75 SC1 464.6 1.80 0.73 SC2 475.1 1.80 0.74

Titration Experiment: Done to determine the optimal concentration of anhydrous tetracycline (ATC) to be used and to determine if the cas9 protein was lethal to the cells. The plan is to make 50 mL of BWF+ w/cas9 and BWF+ w/o cas9 with a starting OD600 of 0.05. These will be placed in an incubator and allowed to grow until reaching 0.5. They will then be diluted again to 0.05 (in 50 mL). Serial dilutions will be made and plated on the prepared plates which will be incubated overnight.
Starting ODs were taken of a 1:10 dilution from liquid cultures grown overnight. This was used to calculate the amount to be added to 50 mL LB to start the experiment with an OD close to 0.05. ODs were taken periodically as the cells grew as shown below.

BWF+ 0.544 460 ?L BWF+ w/cas9 0.556 450 ?L

T0 0.059 0.058 T1 0.081 0.078 T2 0.180 0.168 T3 0.291 0.272 T4 0.398 0.366 T5 0.538 0.489 T6* 0.968 0.874 T7 0.165 0.153 T8 0.414 0.396 T9† 0.561 0.490 T10 0.057 0.056

*At T6, the cultures were again diluted down (5.17 mL BWF+, 5.72 mL BWF+ w/cas9 into 50 mL).
†After T9, 4.898 mL BWF+ and 4.278 mL BWF+ w/ cas9 were again diluted into 50 mL LB. A final OD was taken to ensure that the final OD was close to 0.05 before the cells were plated.
Cells were plated on the previously prepared plates containing increasing concentrations of ATC as previously described (6/9/14) and placed in 37°C incubator overnight.
6/11/14
Titration Experiment: Results
As seen from the images in the table below, the cas9 protein inhibits growth of the cells even prior to removal of repression by the TetR repressor, demonstrating some leaky expression. Increasing ATC concentrations results in increased cas9 expression in the cells as expected.
UCB-induced cc9-titration plates-140611.JPG
Nanodrop of gel extracted (GE) and mini-prep (MP) products as shown below:

GE P51 13.7 1.94 1.18 GE AmilCP PCR 27.3 1.81 1.19 GE cas9 PCR 7.8 1.98 0.62 AmilCP DpnI digest 644.8 1.6 0.65 P50 PCR 493.9 1.8 0.73 GA1 MP 107.9 1.87 1.99 GA2 MP 144.9 1.88 2.11 GA3 MP 168.3 1.92 2.29 GA4 MP 131.1 1.91 1.93 GA5 MP 310.6 1.88 2.34 729-1 125.3 1.88 2.11 729-2 178.3 1.90 2.17 729-3 172.6 1.84 1.81 756-1 420.7 1.87 2.25 756-2 106.8 1.89 1.99 

_t

6/12/14

Transformations done of all 6 gRNA cPEC reactions, P50 (+control), and P51 PCR (-control) Gel extraction of Cas9 done to protocol followed by a nanodrop (results below).

Sample ng/ ?L 260/280 260/230
Cas9 5.5 2.12 0.59
_t
P50 PCR amplification performed using the following amounts:
- 25 ?L Master Mix
- 2.5 ?L of forward primer (10 ?M)
- 2.5 ?L of reverse primer (10 ?M)
- 1.0 ?L DNA (1:50 dilution)
- 19.0 ?L NF H2O
Two samples prepared with different thermocycler settings as follows:

Initial Denature 98°C/30 sec 98°C/2 min Denature 98°C/30 sec 98°C/30 sec Anneal 69°C/30 sec 68°C/30 sec Extend 72°C/3.5 min 72°C/5.5 min

  1. of cycles 34 30

Final Extension 72°C/2 min 72°C/5 min

Overnight cultures prepared on GA1-GA5, 729-1, 729-2, 729-3, and 759-1, 759-2
Colonies taken from SC1 plate for overnight cultures (SC-1, SC-2) with 5 mL LB, 10 ?L Amp (50 ng/mL)
6/13/14
Mini-prep done on O/N cultures SC1-1 and SC1-2 followed by a nanodrop. Remainder of culture was spun down into a pellet and frozen.

SC1-1 30.7 2.02 2.06 SC1-2 32.5 1.94 1.48 

_t

Restriction Digestion done following manufacturer’s protocols on the samples as listed below. All were incubated at 37°C for 1 hour followed by 20 minute heat inactivation at 80°C.

Sample Buffer 3 10X BSA Enzyme 1 Enzyme 2 Enzyme Mix* DNA (200 ng) MQ H2O Total Volume
756-1 2.0 ?L 2.0 ?L 0.5 ?L EagI 0.5 ?L XbaI -- 1.0 ?L 14.0 ?L 20.0 ?L
756-2 2.0 ?L 2.0 ?L 0.5 ?L EagI 0.5 ?L XbaI -- 1.87 ?L 13.13 ?L 20.0 ?L
GA1 2.0 ?L 2.0 ?L -- -- 1.0 ?L 1.85 ?L 13.15 ?L 20.0 ?L
GA2 2.0 ?L 2.0 ?L -- -- 1.0 ?L 1.38 ?L 13.62 ?L 20.0 ?L
GA3 2.0 ?L 2.0 ?L -- -- 1.0 ?L 1.19 ?L 13.81 ?L 20.0 ?L
GA4 2.0 ?L 2.0 ?L -- -- 1.0 ?L 1.53 ?L 13.47 ?L 20.0 ?L
GA5 2.0 ?L 2.0 ?L -- -- 1.0 ?L 0.64 ?L 14.36 ?L 20.0 ?L
P51 2.0 ?L 2.0 ?L -- -- 1.0 ?L 4.0 ?L 11.0 ?L 20.0 ?L
Cas9 2.0 ?L 2.0 ?L -- -- 1.0 ?L 2.0 ?L 13.0 ?L 20.0 ?L
  • Enzyme Mix is a 1:1:23 dilution of EcoRI and BamHI

Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.

Lane Sample
1 2-Log DNA Ladder
2 756-1
3 756-2
4 P51 Digest
5 Sb1C3
6 GA1
7 GA2
8 GA3
9 GA4
10 GA5
11 Cas9 Digest 
UCB-induced cc9-gel-140613.JPG


From the above gel, it does not appear that are products are as expected. A repeat digest is done of GA4 and P50. These new products were run on a 1% agarose gel with reagents as comparisons (5 ?L sample plus 1 ?L loading dye for each) as shown below:

Lane Sample
1 2-Log DNA Ladder
2 GA4
3 P50 Digest
4 Sb1C3
5 CmR1
6 Cas9 PCR
7 Cas9 GE
8 GA4 MP

UCB-induced cc9-gel-02-140613.JPG


6/14/14


Gibson Assembly performed both with and without 2% DMSO. Each 20 ?L reaction contained 10 ?L Gibson Master Mix with the following as listed: Reaction #1 - 6.5 ?L GE Cas9 - 3.5 ?L GE AmilCP

Reaction #2 - 1 ?L P50 PCR Product (1:10) - 5 ?L GE AmilCP - 4 ?L MQ H2O

Reaction #3 - 1 ?L P50 PCR Product (1:10) - 5 ?L GE AmilCP - 4 ?L 1:10 dilution of 2% DMSO Bacterial Transformation: Done according to protocol - GA1-GA3 diluted 1:4 transformed with 2 ?L (100 ?L each) - BWF+ w/Cas9 from S.B. (25 ?L full strength and 25 ?L 1:10 dilution) - BWF+ (25 ?L full strength and 25 ?L 1:10 dilution) - NEB competent cells with P51 (50 ?L) - Competent cells with P51 (50 ?L) for comparison

Week 6

6/15/14

GA colonies picked for overnight cultures from 2 plates (11 total- 3 from Plate 1, 8 from plate 2), placed in 5 mL LB with 170 ng/ ?L Amp.


6/16/14

Mini-prep was done to protocol on all overnight cultures followed by a nanodrop with results listed below:

Sample ng/ ?L 260/280 260/230
1-1 17.5 2.08 1.94
1-2 54.1 1.93 1.96
1-3 30.4 2.00 1.78
2-1 45.8 1.93 1.90
2-2 45.4 1.92 1.89
2-3 46.9 1.93 1.97
2-4 53.5 1.96 2.01
2-5 53.5 1.94 2.04
2-6 41.9 1.95 1.84
2-7 59.3 1.94 1.99
2-8 46.5 1.94 1.98
_t
Next, a restriction digest was performed on all the samples listed above. EcoRV was used in a 1:100 dilution with Buffer D (both from Promega). Samples were incubated for 1 hour at 37°C and heat inactivation performed for 5 minutes at 80°C. The following amounts were used for all the samples except 1-1 (DNA increased to 6.0 ?L and MQ H2O was not added).
- 1.0 ?L Buffer D
- 3.0 ?L Enzyme
- 3.0 ?L sample DNA
- 3.0 ?L MQ H2O
- Total Reaction Volume = 10 ?L
Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.

1 2-Log DNA Ladder 2 1-1 3 1-2 4 1-3 5 2-1 6 2-2 7 2-3 8 2-4 9 2-5 10 2-6 11 2-7 12 2-8

Repeated digestion (protocol above) on select samples to run again for more definition on gel. Samples are run on a 1% agarose gel. The following lists the sample loaded into each lane.

1 2-Log DNA Ladder 2 1-1 3 1-2 4 2-1 5 sb1c3 6 Digested sb1c3 7 Digested p50 8 2-Log DNA Ladder

UCB-induced cc9-gel-140616.JPG
Bacterial Transformation: Done according to protocol
- 7 CPEC products including p51 as the negative control (10 ?L 1:4 dilution) with 40 ?L DH5?
- BWF+ w (40?L)/ S.B. cas9 (1 ?L)
- BWF+ (1 ?L)
Gibson Assembly products 1-1 thru 1-8 and 2-1 thru 2-3 pelleted down and frozen
6/17/14

GA x 1-1 1.0 ?L cutsmart 3.0 ?L XbaI (1:50) -- 3.0 ?L 3.0 ?L 10.0 ?L GA x 1-2 1.0 ?L cutsmart 3.0 ?L XbaI (1:50) -- 3.0  ?L 3.0 ?L 10.0 ?L GA x 2-1 1.0 ?L cutsmart 3.0 ?L XbaI (1:50) -- 3.0 ?L 3.0 ?L 10.0 ?L p50 linear 1.0 ?L cutsmart 3.0 ?L SepI (1:50) -- 3.0 ?L 3.0 ?L 10.0 ?L GA H 1-1 1.0 ?L Fast green digest -- 3.0 ?L 3.0 ?L 3.0 ?L 10.0 ?L GA H 1-2 1.0 ?L Fast green digest -- 3.0 ?L 3.0 ?L 3.0 ?L 10.0 ?L GA H 2-1 1.0 ?L Fast green digest -- 3.0 ?L 3.0 ?L 3.0 ?L 10.0 ?L p50 1.0  ?L Fast green digest -- 3.0 ?L 3.0 ?L 3.0 ?L 10.0 ?L

*Enzyme mix: 1:1:23 dilution of xhoI and HindIII
Digested products are run on a 1% agarose gel. The following lists the sample loaded into each lane.

1 2-Log DNA Ladder 2 GA x 1-1 3 GA x 1-2 4 GA x 2-1 5 p50 6 sb1c3 7 GA H 1-1 8 GA H 1-2 9 GA H 2-1 10 p50 11 2-Log DNA Ladder

UCB-induced cc9-gel-140617.JPG
6/18/14
Colony PCR is used to rapidly screen colonies from cPEC reactions. A colony is pricked using a pipette tip, avoiding scooping up any LB agar since this inhibits the PCR reaction. There are ~10^9 cells are in each colony - just barely touching the colony and transferring a tiny fraction of it is more than sufficient for this reaction. This tip is rubbed into the PCR tube . Tips were then ejected into 3ml LB+AMP for O/N culture.The following was added to each tube to make a 25 ?L reaction.
- 12.5 ?L 2X Master Mix
- 1.25 ?L of forward primer (10 ?M)
- 1.25 ?L of reverse primer (10 ?M)
- 10.0 ?L MQ H2O
For this reaction, the thermocycler was programmed as follows (note the longer time to initially lyse the cells and denature the DNA).

Denature 98°C/30 sec Anneal 65°C/30 sec Extend 72°C/30 sec

  1. of cycles 35

Final Extension 72°C/5 min

6/19/14
Glycerol stocks done to protocol for two colonies from each gRNA that were indicated positive by colony PCR results.
Week 7
6/23/14
Chemically competent cells of SBcas9 • BWF+. Done according to protocol.
6/25/14
Mini prep done to protocol on CPEC products.
Nanodrops were taken to assess the concentration and purity of the samples:

2H 65.2 1.88 2.21 

    3E	56.3	1.91	2.01

4E 38.9 1.93 2.22 5B 54.1 1.86 1.54 6B 69.4 1.88 2.12 7B 66.6 1.82 1.56 8B 69.7 1.83 1.75 9B 61.1 1.89 1.81 10C 52.2 1.86 1.65 11B 43.9 1.86 1.33 12B 57.5 1.83 1.53

Sequencing 12 CPEC products: 12.5 ?L DNA + 2.5 ?L 10?m primer (CRE22)

1 1A 2 1C 3 2B 4 2C 5 3B 6 3H 7 4C 8 4D 9 5E 10 5F 11 6C 12 6F

6/26/14
Sequencing: Total volume 0f 15 ?L

1 2B 12.5?L 2.5 ?L -- 2 2C 12.0?L 2.5?L 0.5?L 3 3B 10.3?L 2.5?L 2.2?L 4 3H 11?L 2.5?L 1.5?L 5 4E 12.5?L 2.5?L -- 6 5E 11.3?L 2.5?L 1.2?L 7 11B 12.5?L 2.5?L -- 8 2H 8?L 2.5?L 4.5?L 9 6B 8?L 2.5?L 4.5?L 10 7B 8?L 2.5?L 4.5?L 11 8B 8?L 2.5?L 4.5?L 12 9B 8?L 2.5?L 4.5?L 13 4C 8?L 2.5?L 4.5?L 14 3E 10?L 2.5?L 2.5?L 15 5B 10?L 2.5?L 2.5?L 16 10C 10?L 2.5?L 2.5?L 17 12B 10?L 2.5?L 2.5?L

Bacterial Transformation of SBcas9• ER: Done according to protocol.
6/27/14
Mini prep done to protocol on CPEC products.
Nanodrops were taken to assess the concentration and purity of the samples:

1A 66.9 1.90 2.26 

    1C	61.3	1.92	2.20

2B 57.8 1.91 2.27 3B 69.4 1.88 1.14 3H 51.7 1.89 2.24 9B 33.6 1.99 1.62 10C 49.8 1.93 2.09 11B 40.5 1.94 2.01 12B 51.9 1.89 1.17

Sequenced all of the above products (total volume of 15 ?L: 10 ?L DNA+ 2.5 ?Lprimer (CRE22)+2 .5 ?L MQ H2O
Exceptions:
-9B received 12.5 ?L DNA + 2.5 ?L primer (CRE22)
-12B received p51 seq1 as the primer
Glycerol stock of all the above samples to protocol: 1:1 w/ 40% glycerol for a total volume of 500 ?L each.
Positive sequencing results: 1A, 1C, 2B, 3B, 3H, 9B, 10C, 11B, 12B
Bacterial Transformation of SBcas9• ER and PUC19 as the positive control: Done according to protocol.
Prepared 5mL O/N cultures in LB+AMP of colonies selected from glycerol stocks
Mini preps done to protocol on the below samples
Nanodrops were taken to assess the concentration and purity of the samples:

1A 78.7 1.90 2.08 

    1C	54.3	1.88	1.76

2B 335.4 1.48 0.91 3B 52.1 1.91 1.96 3H 59.7 1.89 2.03

Week 8
6/30/14
Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51: Done according to protocol.
7/1/14
Nanodrops were taken to assess the concentration of the samples to use in a cPEC reaction.

p50 Amplicon 27.3 

    GE AmilCP	493.9
*Gel extracted
cPEC performed to combine the p50 amplicon with AmilCP following the cPEC protocol and using the following amounts:

Q5 Master Mix 12.5 ?L Insert (AmilCP) 5 ?L Template DNA p50 2.0 ?L (1:20 dilution) 25% DMSO 2.0 ?L Nuclease Free H2O 3.5 ?L Final Reaction Volume 25.0 ?L

Thermocycler conditions:

Initial Denature 98°C/30 sec Denature 98°C/10 sec Anneal 60°C/30 sec Extend 72°C/105 sec

  1. of cycles 10

Final Extension 72°C/10 min

Successful Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51 (from 6/30) . O/N cultures of each were then made.
7/4/14
Electroporation of p50 amplicon • AmilCP with Marcello
7/7/14
Electroporation was unsuccessful. Redone using Bacterial Transformation done to protocol.
Primer design for qPCR
Streaked p50 on plate
Week 9
7/8/14
mini prepped p50 done to protocol
Colony PCR done to protocol on the p50+AmilCP transformation product . Selected 12 colonies.
*PCR tube lids opened in thermocycler for samples: 8, 12, and positive control
Colony PCR products are run on a 1% agarose gel. The following lists the sample loaded into each lane.

1 2-Log DNA Ladder 2 1 3 2 4 3 5 4 6 5 7 6 8 7 9 8 10 9 11 10 12 11 13 12 14 (+) control 15 (-) control

7/9/14
Mini prepped colony PCR samples: 1,7,8,10,11, and p50
Followed by nanodrop with results listed below:

1 89 1.90 2.22 7 110.9 1.88 2.22 8 134.4 1.85 1.77 10 140.9 1.87 2.10 11 93.6 1.89 2.21 p50 191.7 1.87 2.35

The above samples (except 8) were sent for sequencing.
Total volume 0f 15 ?L

1 1 6.0?L 5.0 ?L 4.0?L 2 7 5.0?L 5.0?L 5.0?L 3 10 4.0?L 5.0?L 6.0?L 4 11 6.0?L 5.0?L 4.0?L

Transformation done to protocol on all gRNAs into BWF+
7/10/14
Site directed Mutagenesis (SDM) performed on original p50, 1, 7, 10, 11 to remove Xba1 from p50
Primer names: p50TetRXba1F and p50TetRXba1R
Primer Mix: 1 ?L forward + 1 ?L reverse + 18 ?L MQ H2O
PCR run on the following samples of p50: p50 DNA dilution for original, 1,7,10, and 11
p50 DNA sample original- 1:38 dilution
p50 DNA sample 1- 1:18 dilution
p50 DNA sample 7- 1:22 dilution
p50 DNA sample 10- 1:28 dilution
p50 DNA sample 11- 1:19 dilution

Q5 Master Mix 12.5 ?L 10 ?M primer mix 2.5 ?L DNA p50 2.0 ?L Nuclease Free H2O 8.0 ?L Final Reaction Volume 25.0 ?L

Thermocycler conditions:

Initial Denature 98°C/30 sec Denature 98°C/30 sec Anneal 55°C/30 sec Extend 72°C/3.5 minutes

  1. of cycles 15 cycles

Final Extension 72°C/5 minutes

SDM performed on 6 gRNAs and the original p51 to remove spe1 site
PCR preformed:

Q5 Master Mix 12.5 ?L 10 ?M primer mix 2.5 ?L DNA p51 2.0 ?L Nuclease Free H2O 8.0 ?L Final Reaction Volume 25.0 ?L

*primer mix: 1 ?L forward primer+ 1 ?L reverse primer +18 ?L MQH2O. Primers varied for each gRNA
*control consisted of DNA only, no primers
Thermocycler conditions:

Initial Denature 98 95°C/30 sec Denature 98 95°C/30 sec Anneal 55 55°C/1min Extend 72 68°C/3min

  1. of cycles 15 cycles

Final Extension 72 3 min

7/11/2014
All PCR gRNAs DPN1 digested:
1. Add 1 ?l of the Dpn I restriction enzyme (10 U/?l) directly to each
amplification reaction below the mineral oil overlay.
2. Gently and thoroughly mix each reaction mixture by pipetting the
solution up and down several times. Spin down the reaction mixtures in
a microcentrifuge for 1 minute and immediately incubate each reaction
at 37°C for 1 hour to digest the parental (i.e., the nonmutated)
supercoiled dsDNA.
Transformation done to protocol for all gRNAs into DH5?. Full volume (250?l) plated onto Amp plates and incubated o/n.
Week 10
7/16
O/N cultures of SDM gRNA p51 (4 colonies) and SC1 (2 colonies)
7/17
Mini prepped all p51 and SC1 cultures to protocol and then nano dropped:

p51 (1) 62.5 1.90 1.65 

   p51(2)	87.2	1.95	2.20

p51(3) 112.7 1.81 1.34 p51(4) 67.1 1.92 2.24 sc1 (1) 126.5 1.83 1.48 sc1(2) 101.9 1.88 1.84

Restriction digest done to protocol:

(200 ng) MQ H2O Total Volume p51 (1) 1.0 ?L 1.0 ?L 3.0 ?L 5.0 ?L 10.0 ?L p51 (2) 1.0 ?L 1.0 ?L 2.0 ?L 6.0 ?L 10.0 ?L p51 (3) 1.0 ?L 1.0 ?L 2.0 ?L 6.0 ?L 10.0 ?L p51 (4) 1.0 ?L 1.0 ?L 3.0 ?L 5.0 ?L 10.0 ?L SC1 (1) 1.0 ?L 1.0 ?L 2.0 ?L 6.0 ?L 10.0 ?L SC1 (2) 1.0 ?L 1.0 ?L 2.0 ?L 6.0 ?L 10.0 ?L

*Enzyme Mix contains 1.0 ?L Spe1 + 1.0 ?L BgI1 + 23.0 ?L mqH2O
Samples evaporated after digesting to a total volume of around 4 ?L
Run on a 1% Gel. The following lists the sample loaded into each lane.

1 2-Log DNA Ladder 2 SDM p51 (1) 3 SDM p51 (3) 4 SDM SC1 (1) 5 SDM SC1 (2) 6 Blank 7 SDM P51 (4)

UCB-induced cc9-gel-140717.JPG
7/19/2014
PCR amplify p50

Q5 Master Mix 12.5 ?L 10 ?M forward primer 1.5 ?L 10 ?M reverse primer 1.5 ?L DNA p50 (1:50) 1.0 ?L Nuclease Free H2O 9.0 ?L Final Reaction Volume 25.0 ?L

7/21/2014
Restriction digest same gRNAs again

(200 ng) MQ H2O Total Volume p51 (1) 2.0 ?L 1.0 ?L 1.0 ?L 3.2 ?L 12.8 ?L 20.0 ?L p51 (2) 2.0 ?L 1.0 ?L 1.0 ?L 2.3 ?L 13.7 ?L 20.0 ?L p51 (3) 2.0 ?L 1.0 ?L 1.0 ?L 1.8 ?L 14.2 ?L 20.0 ?L p51 (4) 2.0 ?L 1.0 ?L 1.0 ?L 3.0 ?L 13.0?L 20.0 ?L SC1 (1) 2.0 ?L 1.0 ?L 1.0 ?L 1.6 ?L 14.4 ?L 20.0 ?L SC1 (2) 2.0 ?L 1.0 ?L 1.0 ?L 2.0 ?L 14.0?L 20.0 ?L P51 2.0 ?L 1.0 ?L 1.0 ?L 4.0 ?L 12.0?L P51 2.0 ?L 1.0 ?L 1.0 ?L 4.0 ?L 12.0?L p51 2.0 ?L 1.0 ?L 1.0 ?L 4.0 ?L 12.0?L

Enzymes are used as a 1:25 dilution
Run on a 1% Gel. The results were still unclear, the following day samples were sent for sequencing.
Week 11
7/23/2014
Sent p51 SDM 1, 3, 4 and p51 original as a control for sequencing. Confirmation that SDM was successful and p51 no longer has the restriction site spe1 in the gRNA location.
Sequencing: Total volume 0f 15 ?L

1 SDM1p51 8.0?L 2.5 ?L 4.5?L 2 SDM3p51 4.4?L 2.5?L 8.1?L 3 SDM4p51 7.5?L 2.5?L 5.0?L 4 p51 control 4.2?L 2.5?L 8.3?L

Mini prepped 10 o/n cultures of SDM of p50. Done to protocol. Glycerol stocks make of all 10 cultures. Mini prepped previous gibson colonies 1 and 2. SDM of p51 for all samples were confirmed
Nanodrops were taken to assess the concentration and purity of the samples:

p50 123.7 1.84 2.15 

    SDM1	179.6	1.88	2.19

SDM2 117.4 1.90 2.21 SDM3 140.4 1.84 1.81 

   SDM4	162.6	1.89	2.22

SDM5 117.3 1.89 2.00 SDM6 176.4 1.88 2.01 SDM7 177.0 1.87 2.30 SDM8 250.1 1.89 2.25 SDM9 298.3 1.90 2.31 SDM10 136.0 1.91 2.17 Gibson1 110.8 1.89 2.14 Gibson2 160.3 1.87 2.15 

_t

Restriction digest done to protocol:

Sample Buffer Cutsmart Enzyme Mix* DNA (200ng) MQ H2O Total Volume
SDM p50 (1) 2.0 ?L 1.0 ?L 1.2 ?L 15.8 ?L 20.0 ?L
SDM p50 (2) 2.0 ?L 1.0 ?L 2.0 ?L 15.0 ?L 20.0 ?L
SDM p50 (3) 2.0 ?L 1.0 ?L 1.4 ?L 15.6 ?L 20.0 ?L
SDM p50 (4) 2.0 ?L 1.0 ?L 1.2 ?L 15.8?L 20.0 ?L
SDM p50 (5) 2.0 ?L 1.0 ?L 2.0 ?L 15.0 ?L 20.0 ?L
SDM p50 (6) 2.0 ?L 1.0 ?L 1.2 ?L 15.8 ?L 20.0 ?L
SDM p50 (7) 2.0 ?L 1.0 ?L 1.2 ?L 15.8?L 20.0 ?L
SDM p50 (8) 2.0 ?L 1.0 ?L 1.0 ?L 16.0?L 20.0 ?L
SDM p50 (9) 2.0 ?L 1.0 ?L 1.0 ?L 16.0?L 20.0 ?L
SDM p50 (10) 2.0 ?L 1.0 ?L 1.5 ?L 15.5?L 20.0 ?L
p50control 2.0 ?L 1.0 ?L 2.0 ?L 15.0?L 20.0 ?L
  • Enzyme Mix contains 1.0 ?L Spe1 + 1.0 ?L Xba1 + 48.0 ?L mqH2O

Restriction Digest for gibson done to protocol:

Sample Buffer D Enzyme EcoRV DNA
(200 ng) MQ H2O Total
Volume
Gibson 1 2.0 ?L 1.0 ?L 2.0 ?L 15.0 ?L 20.0 ?L
Gibson 2 2.0 ?L 1.0 ?L 1.2 ?L 15.8 ?L 20.0 ?L
  • EcoRv dilution (1:25)


Run on a 1% Gel. The following lists the sample loaded into each lane.

Lane Sample
1 2-Log DNA Ladder
2 SDM p50 (1)
3 SDM p50 (2)
4 SDM p50 (3)
5 SDM p50 (4)
6 SDM p50 (5)
7 SDM p50 (6)
8 SDM p50 (7)
9 SDM p50 (8)
10 SDM p50 (9)
11 SDM p50 (10)
12 p50 control
13 p50 undigested
14 Gibson 1
15 Gibson 2
16 sb1c3
17 blank

UCB-induced cc9-gel-140723.JPG


7/25/2014

Restriction digest done to protocol:

Sample Buffer Cutsmart Enzyme Mix* DNA
(250 ng) MQ H2O Total
Volume
SDM p50 (1) 2.0 ?L 1.0 ?L 1.5 ?L 20.0 ?L 25.0 ?L
SDM p50 (2) 2.0 ?L 1.0 ?L 2.0 ?L 19.5 ?L 25.0 ?L
SDM p50 (3) 2.0 ?L 1.0 ?L 1.8 ?L 19.7 ?L 25.0 ?L
SDM p50 (4) 2.0 ?L 1.0 ?L 1.5 ?L 20.0?L 25.0 ?L
SDM p50 (5) 2.0 ?L 1.0 ?L 2.0 ?L 19.5 ?L 25.0 ?L
SDM p50 (6) 2.0 ?L 1.0 ?L 1.5 ?L 20.0 ?L 25.0 ?L
SDM p50 (10) 2.0 ?L 1.0 ?L 2.0 ?L 19.5?L 25.0 ?L
p50control 2.0 ?L 1.0 ?L 2.0 ?L 19.5?L 25.0 ?L
  • Enzyme mix: 5.5?L Xba1 + 5.5?L Spe1 for a total volume of 11?L

Run on a 1% Gel. Results were not as anticipated. Restriction Ligation was repeated.

Restriction Ligation

Sample Buffer Cutsmart EcoR1 Xba1 Spe1 Pst1 DNA
(200 ng) MQ H2O Total
Volume
SDM p50 (1) 5.0 ?L -- 1.0 ?L -- 1.0 ?L 1.0 ?L 42.0 ?L 50.0 ?L
SDM p50 (4) 5.0 ?L -- 1.0 ?L -- 1.0 ?L 1.0 ?L 42.0 ?L 50.0 ?L
SDM p50 (6) 5.0 ?L -- 1.0 ?L -- 1.0 ?L 1.0 ?L 42.0 ?L 50.0 ?L
Litimus28i 5.0 ?L -- -- 1.0 ?L 1.0 ?L 1.5 ?L 41.5?L 50.0 ?L
Litimus+Ic3 5.0 ?L 1.0 ?L -- -- 1.0 ?L 0.8 ?L 42.2 ?L 50.0 ?L
PsBGA1 (RFP) 5.0 ?L 1.0 ?L -- 1.0 ?L -- 3.8 ?L 39.2 ?L 50.0 ?L

Sequenced SDMp50 was not the correct product, the above project did not continue.

8/18/2014 O/N cultures of MG, Bw+Kan, Bw-kan

8/19/2014 O/N cultures were all made competent according to protocol O/D recordings:

Time MG Bw+kan Bw(-)kan
11:00 .042 .026 .071
11:34 .086 .035 .088
12:10 .239 .078 .223
12:20 .328 .098 .296
12:28 -- -- .325
12:47 -- .196 --
1:10 -- .308 --
1:15 -- .320 --

The cells were all pulled at the time they were at an O/D nearest .325. All O/D’s were diluted to .30 Once the cells were made competent they were transformed using a gRNA target, gRNA scramble, and a sb1c3 strain.

Retrieved from "http://2014.igem.org/Team:CU-Boulder/Notebook/CC9_Team/Induced"