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From 2014.igem.org

Contents 1 Week 1 2 Week 2 3 Week 3 4 Week 4 5 Week 5 6 Week 6 7 Week 7 8 Week 8 9 Week 9 10 Week 10 11 Week 11

Week 1

5/12

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

Glycerol stocks - Stocks made from overnight cultures (3 from each) - 500 uL bacteria + 500 uL 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

Nano Drop of Mini Prep
Sample	260/280 Ratio	260/230 Ratio	DNA Concentration
P44250	1.94	2.17	49.4 ng/uL
P44251	1.94	2.47	52.7 ng/uL

• 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 uL	5.0 uL
XbaI	1.0 uL	1.0 uL
XhoI	1.0 uL	--
SpeI	1.0 uL	--
EcoRI-HF	--	1.0 uL
DNA (200 ng)	4.0 uL	4.0 uL
MQ H2O	38.0 uL	39.0 uL
Total Volume	50.0 uL	50.0 uL

Agarose Gel Electrophoresis -1% agarose gel prepared -2 uL loading dye added to 18 uL 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.

Plate Streaking

-Used to check the TetR gene in the P44250 plasmid

-2 mL LB containing 50 mg/ uL 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

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.

5/16

Dry Lab

-gRNA designs modified

-Primer design for Gibson assembly of our plasmid

Week 2

5/19

Transformation:

1 uL 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 (u plate with 1:10 dilution, u plate 1:1), Amp + Kan, Amp + Kan (u plate with 1:10 dilution, u plate 1:1)

5/20

Plates from 5/19/14 (shown below).

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

Transformation: (Chemically competent BWF+ cells with P44250 plasmid)

1 uL P44250 (cas9) plasmid added to conjugated BWF+ strain

Transformation protocol followed with following exceptions:

-200 uL SOC added to each sample

-Incubated for 2 hours

-1:10 dilution made (10 uL into 90 uL SOC)

-Cultures plated on Amp plates, one with full concentration and another with the 1:10 dilution

5/23

Arrival of primers previously ordered

All were reconstituted in MQ H2O to 100 uM 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)

Single colonies taken from the BFW+ w/ P50 plasmid plate in the 1:10 dilution and placed in 2 uL liquid culture overnight with the following antibiotics:

-AMP

-Kan

-AMP + Kan

-Tet

5/24

As shown the image below, overnight cultures from 5/23/14 are all cloudy demonstrating bacterial growth.

PCR Reaction – Used to amplify out cas9 from P44250 plasmid and CMR from PSB1C3 plasmid

• DNA samples diluted so each contains 0.5 ng/uL

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

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

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 uL EtBr.

Lane 1: 5 uL 2-Log DNA Ladder

Lane 2: CMR sample* (~2 Kb size)

Lane 3: Cas9 sample* (~5 Kb size)

• Each contained 8 uL reaction sample + 2 uL 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).

5/27

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 uL
dCMR Primer	2.5 uL
Q5 Polymerase	0.5 uL
CMR DNA	2.0 uL
Nuclease Free H2O	20 uL
Final Reaction Volume	50.0 uL

5/29

Gel electrophoresis: 1% gel ran at 110V for ~40 minutes.

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

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)

Gel extraction performed on cas9, CMR1, and CMR2 lanes using E.Z.N.A. Gel extraction kit following the protocol. Following amounts:

-Cas9: 110 uL buffer with 0.1103 g sample

-CMR1: 53 uL buffer with 0.0533 g sample

-CMR2: 90 uL buffer with 0.0904 g sample

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

Sample	ng/ uL	260/280	260/230	**ng/ uL	**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 uL samples

-25 uL binding buffer

-Washed 2x with 700 uL SPW

-30 uL elution buffer added, allowed to rest 4 minutes

-Centrifuged on max for 2 min

5/30

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 uL sample

1.0 uL DPN1 (100 fold dilution)

3.0 uL 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 uL
Cas9	1.6 uL (50 ng)
Cmr	2.4 uL (143 ng)
Nuclease Free H2O	6 uL
Final Reaction Volume	20.0 uL

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

Week 4

6/2

Grow Gibson colonies in 6 mL LB media + 30 uL chloroamp

Transform Gibson colonies following protocol and with amounts as shown:

-BWF+ w/cas9: 15 uL into 135 uL SOC

-BW w/cas9: 15 uL into 135 uL SOC

-BWF+ (no cas9): 30 uL into 270 uL SOC

-BW (no cas9): 30 uL into 270 uL SOC

90 uL 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

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/ uL	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 uL	2.5 uL
EcoRV	0.5 uL	0.5 uL
BamHI	0.5 uL	0.5 uL
BSA (1:4 dilution)	1.0 uL	1.0 uL
DNA (250 ng)	1.25 uL	5.0 uL
MQ H2O	19.25 uL	15.5 uL
Total Volume	25.0 uL	25.0 uL

1% Gel made for electrophoresis. Each sample had 1 uL of loading dye added, 5 uL of DNA ladder and 5 uL 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.

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 uL
CRE1 Primer	2.5 uL
CRE4 Primer	2.5 uL
P51 DNA	2.0 uL
Q5 Polymerase	0.5 uL
Nuclease Free H2O	17.5 uL
Final Reaction Volume	50.0 uL

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 uL sample

1.0uL DPN1 (100 fold dilution)

3.0 uL 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 uL
Primer	1.7 uL
Primer	2.0 uL
Q5 Polymerase	0. 5 uL
Nuclease Free H2O	19.5 uL
Final Reaction Volume	50.0 uL

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/ uL	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 uL
Insert (our primer dimers)	1.7 uL
Template DNA	2.0 uL
Q5 Polymerase	0.25 uL
Nuclease Free H2O	10.05 uL
Final Reaction Volume	25.0 uL

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 uL
Cas9	1.6 uL (50 ng)
Cmr	2.4 uL (143 ng)
Nuclease Free H2O	6 uL
Final Reaction Volume	20.0 uL

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 uL Kan + 10 uL Amp
BW w/ cas	5 uL Kan + 10 uL Amp
BWF+	5 uL Kan
BW	5 uL Kan
BWF+ (not transformed)	5 uL Kan + 10 uL Tet

6/4

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

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 uL
Cas9 DNA	5.0 uL
CmR DNA	5.0 uL
Nuclease Free H2O	0.00 uL
Final Reaction Volume	20.0 uL

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

Nanodrop done on the three cPEC casC reactions with results listed below:

Sample	ng/ uL	260/280	260/230
CasC1	489.3	1.80	0.72
CasC2	577.7	1.76	0.59
CasC3	542.5	1.80	0.74

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

Sample	ng/ uL	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

1% Gel made for electrophoresis. Each sample had 1 uL of loading dye added, 5 uL of DNA ladder and 5 uL of each sample was loaded. Gel ran 122V for ~50 min.

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

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

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).

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

Week 5

6/9

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/ uL	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

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 u containing Kan and u 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 uL	1.25 uL
BxsI	1.0 uL	2.5 uL
DNA (125 ng)	4.0 uL	1.25 uL
Nuclease Free H2O	6.25 uL	7.5 uL
Final Reaction Volume	12.5 uL	12.5 uL

6/10

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 uL of each to 40 uL nuclease free H2O (N.F. H2O). Two separate primer mixes were made (one for p51 and one for AmilCP) using 5 uL of each forward and reverse primer dimer that were added to 40 uL of N.F. H2O to produce a dilution (1:1:8). 5 uL of each primer mix was added to 25 uL of the Q5 master mix and 20 uL N.F. H2O to make a 50 uL total reaction for each sample.Products were checked by gel electrophoresis (shown below).

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 uL
Primer Mix	2.5 uL
DNA (1:10 dilution)	1.0 uL
Nuclease Free H2O	25.5 uL
Final Reaction Volume	54.0 uL

Following the cPEC reactions, Nanodrops were taken to assess the concentration and purity of the samples:

Sample	ng/ uL	260/280	260/230
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.

Sample	OD	Amount added to 50 mL
BWF+	0.544	460 uL
BWF+ w/cas9	0.556	450 uL

Time	BWF+	BWF+ w/cas9
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

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.

Nanodrop of gel extracted (GE) and mini-prep (MP) products as shown below:

Sample	ng/ uL	260/280	260/230
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

6/12

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/ uL	260/280	260/230
Cas9	5.5	2.12	0.59

P50 PCR amplification performed using the following amounts:

-25 uL Master Mix

-2.5 uL of forward primer (10 uM)

-2.5 uL of reverse primer (10 uM)

-1.0 uL DNA (1:50 dilution)

-19.0 uL NF H2O

Two samples prepared with different thermocycler settings as follows:

	1st Round	2nd Round
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
# 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 uL Amp (50 ng/mL)

6/13

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.

Sample	ng/ uL	260/280	260/230
SC1-1	30.7	2.02	2.06
SC1-2	32.5	1.94	1.48

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 uL	2.0 uL	0.5 uL EagI	0.5 uL XbaI	--	1.0 uL	14.0 uL	20.0 uL
756-2	2.0 uL	2.0 uL	0.5 uL EagI	0.5 uL XbaI	--	1.87 uL	13.13 uL	20.0 uL
GA1	2.0 uL	2.0 uL	--	--	1.0 uL	1.85 uL	13.15 uL	20.0 uL
GA2	2.0 uL	2.0 uL	--	--	1.0 uL	1.38 uL	13.62 uL	20.0 uL
GA3	2.0 uL	2.0 uL	--	--	1.0 uL	1.19 uL	13.81 uL	20.0 uL
GA4	2.0 uL	2.0 uL	--	--	1.0 uL	1.53 uL	13.47 uL	20.0 uL
GA5	2.0 uL	2.0 uL	--	--	1.0 uL	0.64 uL	14.36 uL	20.0 uL
P51	2.0 uL	2.0 uL	--	--	1.0 uL	4.0 uL	11.0 uL	20.0 uL
Cas9	2.0 uL	2.0 uL	--	--	1.0 uL	2.0 uL	13.0 uL	20.0 uL

• 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

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 uL sample plus 1 uL 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

6/14

Gibson Assembly performed both with and without 2% DMSO. Each 20 uL reaction contained 10 uL Gibson Master Mix with the following as listed:

Reaction #1

-6.5 uL GE Cas9

-3.5 uL GE AmilCP

Reaction #2

-1 uL P50 PCR Product (1:10)

-5 uL GE AmilCP

-4 uL MQ H2O

Reaction #3

-1 uL P50 PCR Product (1:10)

-5 uL GE AmilCP

-4 uL 1:10 dilution of 2% DMSO

Bacterial Transformation: Done according to protocol

-GA1-GA3 diluted 1:4 transformed with 2 uL (100 uL each)

-BWF+ w/Cas9 from S.B. (25 uL full strength and 25 uL 1:10 dilution)

-BWF+ (25 uL full strength and 25 uL 1:10 dilution)

-NEB competent cells with P51 (50 uL)

-Competent cells with P51 (50 uL) for comparison

Week 6

6/15

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/ uL Amp.

6/16

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

Sample	ng/ uL	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

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 uL and MQ H2O was not added).

-1.0 uL Buffer D

-3.0 uL Enzyme

-3.0 uL sample DNA

-3.0 uL MQ H2O

-Total Reaction Volume = 10 uL

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	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.

Lane	Sample
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

Bacterial Transformation: Done according to protocol

-7 CPEC products including p51 as the negative control (10 uL 1:4 dilution) with 40 uL DH5u

-BWF+ w (40uL)/ S.B. cas9 (1 uL)

-BWF+ (1 uL)

Gibson Assembly products 1-1 thru 1-8 and 2-1 thru 2-3 pelleted down and frozen

6/17

Sample	Buffer	Enzyme 1	EnzymeMix*	DNA (200 ng)	MQ H2O	Total Volume
GA x 1-1	1.0 uL cutsmart	3.0 uL XbaI (1:50)	--	3.0 uL	3.0 uL	10.0 uL
GA x 1-2	1.0 uL cutsmart	3.0 uL XbaI (1:50)	--	3.0 uL	3.0 uL	10.0 uL
GA x 2-1	1.0 uL cutsmart	3.0 uL XbaI (1:50)	--	3.0 uL	3.0 uL	10.0 uL
p50 linear	1.0 uL cutsmart	3.0 uL SepI (1:50)	--	3.0 uL	3.0 uL	10.0 uL
GA H 1-1	1.0 uL Fast green digest	--	3.0 uL	3.0 uL	3.0 uL	10.0 uL
GA H 1-2	1.0 uL Fast green digest	--	3.0 uL	3.0 uL	3.0 uL	10.0 uL
GA H 2-1	1.0 uL Fast green digest	--	3.0 uL	3.0 uL	3.0 uL	10.0 uL
p50	1.0 uL Fast green digest	--	3.0 uL	3.0 uL	3.0 uL	10.0 uL

• 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.

Lane	Sample
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

6/18

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 uL reaction.

-12.5 uL 2X Master Mix

-1.25 uL of forward primer (10 uM)

-1.25 uL of reverse primer (10 uM)

-10.0 uL MQ H2O

For this reaction, the thermocycler was programmed as follows (note the longer time to initially lyse the cells and denature the DNA).

Initial Denature	98°C/10 min
Denature	98°C/30 sec
Anneal	65°C/30 sec
Extend	72°C/30 sec
# of cycles	35
Final Extension	72°C/5 min

6/19

Glycerol stocks done to protocol for two colonies from each gRNA that were indicated positive by colony PCR results.

Week 7

6/23

Chemically competent cells of SBcas9 • BWF+. Done according to protocol.

6/25

Mini prep done to protocol on CPEC products.

Nanodrops were taken to assess the concentration and purity of the samples:

Sample	ng/ uL	260/280	260/230
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 uL DNA + 2.5 uL 10um primer (CRE22)

	CPEC product
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

Sequencing: Total volume 0f 15 uL

	CPEC Product	DNA uL	Primer CRE22	MQ H2O
1	2B	12.5uL	2.5 uL	--
2	2C	12.0uL	2.5uL	0.5uL
3	3B	10.3uL	2.5uL	2.2uL
4	3H	11uL	2.5uL	1.5uL
5	4E	12.5uL	2.5uL	--
6	5E	11.3uL	2.5uL	1.2uL
7	11B	12.5uL	2.5uL	--
8	2H	8uL	2.5uL	4.5uL
9	6B	8uL	2.5uL	4.5uL
10	7B	8uL	2.5uL	4.5uL
11	8B	8uL	2.5uL	4.5uL
12	9B	8uL	2.5uL	4.5uL
13	4C	8uL	2.5uL	4.5uL
14	3E	10uL	2.5uL	2.5uL
15	5B	10uL	2.5uL	2.5uL
16	10C	10uL	2.5uL	2.5uL
17	12B	10uL	2.5uL	2.5uL

Bacterial Transformation of SBcas9• ER: Done according to protocol.

6/27

Mini prep done to protocol on CPEC products. Nanodrops were taken to assess the concentration and purity of the samples:

Sample	ng/ uL	260/280	260/230
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 uL: 10 uL DNA+ 2.5 uLprimer (CRE22)+2 .5 uL MQ H2O Exceptions:

-9B received 12.5 uL DNA + 2.5 uL 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 uL 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:

Sample	ng/ uL	260/280	260/230
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 Bacterial Transformation of BWF+ w/ 158, 723, SC1, p51: Done according to protocol.

7/1

Nanodrops were taken to assess the concentration of the samples to use in a cPEC reaction.

Sample	ng/ uL
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:

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

Thermocycler conditions:

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

Electroporation of p50 amplicon • AmilCP with Marcello

7/7

Electroporation was unsuccessful. Redone using Bacterial Transformation done to protocol.

Primer design for qPCR

Streaked p50 on plate

Week 9

7/8

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.

Lane	Sample
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

Mini prepped colony PCR samples: 1,7,8,10,11, and p50

Followed by nanodrop with results listed below:

Sample	ng/ uL	260/280	260/230
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 uL

	Product	DNA uL	Primer CRE22	MQ H2O
1	1	6.0uL	5.0 uL	4.0uL
2	7	5.0uL	5.0uL	5.0uL
3	10	4.0uL	5.0uL	6.0uL
4	11	6.0uL	5.0uL	4.0uL

Transformation done to protocol on all gRNAs into BWF+

7/10

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 uL forward + 1 uL reverse + 18 uL 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

Reactants	Volume
Q5 Master Mix	12.5 uL
10 uM primer mix	2.5 uL
DNA p50	2.0 uL
Nuclease Free H2O	8.0 uL
Final Reaction Volume	25.0 uL

Thermocycler conditions:

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

Reactants	Volume
Q5 Master Mix	12.5 uL
10 uM primer mix	2.5 uL
DNA p51	2.0 uL
Nuclease Free H2O	8.0 uL
Final Reaction Volume	25.0 uL

• primer mix: 1 uL forward primer+ 1 uL reverse primer +18 uL 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
# of cycles	15 cycles
Final Extension 72	3 min

7/11/2014

All PCR gRNAs DPN1 digested:

1. Add 1 ul of the Dpn I restriction enzyme (10 U/ul) 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 DH5u. Full volume (250ul) 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:

Sample	ng/ uL	260/280	260/230
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:

Sample	Buffer Green Fast Digest	Enzyme Mix*	DNA	MQ H2O	Total Volume
p51 (1)	1.0 uL	1.0 uL	3.0 uL	5.0 uL	10.0 uL
p51 (2)	1.0 uL	1.0 uL	2.0 uL	6.0 uL	10.0 uL
p51 (3)	1.0 uL	1.0 uL	2.0 uL	6.0 uL	10.0 uL
p51 (4)	1.0 uL	1.0 uL	3.0 uL	5.0 uL	10.0 uL
SC1 (1)	1.0 uL	1.0 uL	2.0 uL	6.0 uL	10.0 uL
SC1 (2)	1.0 uL	1.0 uL	2.0 uL	6.0 uL	10.0 uL

• Enzyme Mix contains 1.0 uL Spe1 + 1.0 uL BgI1 + 23.0 uL mqH2O

Samples evaporated after digesting to a total volume of around 4 uL

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

Lane	Sample
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)

7/19 PCR amplify p50

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

7/21

Restriction digest same gRNAs again

Sample	Buffer cutsmart	Enzyme Xbal	Enzyme Spe1	DNA
(200 ng)	MQ H2O	Total
Volume
p51 (1)	2.0 uL	1.0 uL	1.0 uL	3.2 uL	12.8 uL	20.0 uL
p51 (2)	2.0 uL	1.0 uL	1.0 uL	2.3 uL	13.7 uL	20.0 uL
p51 (3)	2.0 uL	1.0 uL	1.0 uL	1.8 uL	14.2 uL	20.0 uL
p51 (4)	2.0 uL	1.0 uL	1.0 uL	3.0 uL	13.0uL	20.0 uL
SC1 (1)	2.0 uL	1.0 uL	1.0 uL	1.6 uL	14.4 uL	20.0 uL
SC1 (2)	2.0 uL	1.0 uL	1.0 uL	2.0 uL	14.0uL	20.0 uL
P51	2.0 uL	1.0 uL	1.0 uL	4.0 uL	12.0uL
P51	2.0 uL	1.0 uL	1.0 uL	4.0 uL	12.0uL
p51	2.0 uL	1.0 uL	1.0 uL	4.0 uL	12.0uL

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

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 uL

	SDM Product	DNA uL	Primer p51seq5	MQ H2O
1	SDM1p51	8.0uL	2.5 uL	4.5uL
2	SDM3p51	4.4uL	2.5uL	8.1uL
3	SDM4p51	7.5uL	2.5uL	5.0uL
4	p51 control	4.2uL	2.5uL	8.3uL

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:

Sample	ng/ uL	260/280	260/230
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

Restriction digest done to protocol:

Sample	Buffer Cutsmart	Enzyme Mix*	DNA (200ng)	MQ H2O	Total Volume
SDM p50 (1)	2.0 uL	1.0 uL	1.2 uL	15.8 uL	20.0 uL
SDM p50 (2)	2.0 uL	1.0 uL	2.0 uL	15.0 uL	20.0 uL
SDM p50 (3)	2.0 uL	1.0 uL	1.4 uL	15.6 uL	20.0 uL
SDM p50 (4)	2.0 uL	1.0 uL	1.2 uL	15.8uL	20.0 uL
SDM p50 (5)	2.0 uL	1.0 uL	2.0 uL	15.0 uL	20.0 uL
SDM p50 (6)	2.0 uL	1.0 uL	1.2 uL	15.8 uL	20.0 uL
SDM p50 (7)	2.0 uL	1.0 uL	1.2 uL	15.8uL	20.0 uL
SDM p50 (8)	2.0 uL	1.0 uL	1.0 uL	16.0uL	20.0 uL
SDM p50 (9)	2.0 uL	1.0 uL	1.0 uL	16.0uL	20.0 uL
SDM p50 (10)	2.0 uL	1.0 uL	1.5 uL	15.5uL	20.0 uL
p50control	2.0 uL	1.0 uL	2.0 uL	15.0uL	20.0 uL

• Enzyme Mix contains 1.0 uL Spe1 + 1.0 uL Xba1 + 48.0 uL mqH2O

Restriction Digest for gibson done to protocol:

Sample	Buffer D	Enzyme EcoRV	DNA
(200 ng)	MQ H2O	Total
Volume
Gibson 1	2.0 uL	1.0 uL	2.0 uL	15.0 uL	20.0 uL
Gibson 2	2.0 uL	1.0 uL	1.2 uL	15.8 uL	20.0 uL

• 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

7/25

Restriction digest done to protocol:

Sample	Buffer Cutsmart	Enzyme Mix*	DNA
(250 ng)	MQ H2O	Total
Volume
SDM p50 (1)	2.0 uL	1.0 uL	1.5 uL	20.0 uL	25.0 uL
SDM p50 (2)	2.0 uL	1.0 uL	2.0 uL	19.5 uL	25.0 uL
SDM p50 (3)	2.0 uL	1.0 uL	1.8 uL	19.7 uL	25.0 uL
SDM p50 (4)	2.0 uL	1.0 uL	1.5 uL	20.0uL	25.0 uL
SDM p50 (5)	2.0 uL	1.0 uL	2.0 uL	19.5 uL	25.0 uL
SDM p50 (6)	2.0 uL	1.0 uL	1.5 uL	20.0 uL	25.0 uL
SDM p50 (10)	2.0 uL	1.0 uL	2.0 uL	19.5uL	25.0 uL
p50control	2.0 uL	1.0 uL	2.0 uL	19.5uL	25.0 uL

• Enzyme mix: 5.5uL Xba1 + 5.5uL Spe1 for a total volume of 11uL

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 uL	--	1.0 uL	--	1.0 uL	1.0 uL	42.0 uL	50.0 uL
SDM p50 (4)	5.0 uL	--	1.0 uL	--	1.0 uL	1.0 uL	42.0 uL	50.0 uL
SDM p50 (6)	5.0 uL	--	1.0 uL	--	1.0 uL	1.0 uL	42.0 uL	50.0 uL
Litimus28i	5.0 uL	--	--	1.0 uL	1.0 uL	1.5 uL	41.5uL	50.0 uL
Litimus+Ic3	5.0 uL	1.0 uL	--	--	1.0 uL	0.8 uL	42.2 uL	50.0 uL
PsBGA1 (RFP)	5.0 uL	1.0 uL	--	1.0 uL	--	3.8 uL	39.2 uL	50.0 uL

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

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

8/19 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.