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Revision as of 22:49, 17 October 2014
Restriction Digest of PCR Product with XbaI and PstI
Thursday, July 3, 2014
Result
1. 8.6 ug/mL for RBS PCR product
2. 11.5 ug/mL for no RBS PCR product
Mini-Prep
Tuesday, July 8, 2014
Mini-prepped 5x5 mL of E. coli dam- pBRES36a in LB+amp
Ran out of lysis solution ,unable to mini prep the remaining 25 mL
Yield:
5x50 mL = 250 mL
Concentration: 42 ug/mL, equivalent to10.5 ng plasmid
Testing old Tubes and Preparing LB Agar (100mL)
Wednesday, July 9, 2014
Testing Old Mini-prep Columns and Tubes:
Use 20 uL of 3.0 ug/mL DNA
Result
New tubes: 52 ug/mL
Old Tubes: 20 ug/mL
Conclusion: old tubes can no longer be used purchased new tubes
100 mL LB Agar and autoclaved:
1g tryptone
0.5g yeast extract
1g NaCl
1.5 g Agar (1.5%)
100 mL Water (ddH2O)
50 mL aliquots
Ligating Hsp60 Inserts Into Vector Backbone
Monday, July 14, 2014
1. Digestion
Materials:
Reagent |
Amount (ul) |
DNA |
40 |
Buffer |
5 |
XbaI restriction enzyme |
1.5 |
PstI restriction enzyme |
1.5 |
ddH2O |
2 |
Total |
50 |
Procedures:
1. Mixed materials together in microcentrifuge tubes
2. Incubate 30 minutes at 37 degrees Celsius
2. PCR Purification (using Promega Wizard SV gel clean up kit)
3. Ligations
a. Ligation 1: RBS and Vector
b. Ligation 2: no RBS and Vector
Materials for Ligation 1:
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of RBS insert |
4 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
9.5 |
Materials for Ligation 2
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of no RBS insert |
3 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
10.5 |
Procedures:
1. 15 minutes at room temperature
2. Put on ice until transformation
4. Transformation (see transformation protocol)
Housekeeping
Monday, July 14, 2014
30 mL LB Agar and autoclaved:
0.3 g tryptone
0.18 g yeast extract
0.3 g NaCl
0.45 g Agar (1.5%)
30 mL Water (ddH2O)
Chloramphenicol (CAM)
10 mg in 1 mL of 95% EtOH
5ug/mL workign concentration is needed
Used 35 ug/mL final
Prep 50 mL LB agar +CAM:
0.5 g tryptone
0.25 g yeast extract
0.5 g NaCl
0.75 g Agar (1.5%)
48 mL Water (ddH2O)
25 uL of 10 mg/mL CAM
Competent cells growing
Housekeeping and Ligation
Wednesday, July 16, 2014
Prep 75 m
0.75 g tryptone
0.375 g yeast extract
0.5 g NaCl
1.125 g Agar (1.5%)
50 mL Water (ddH2O)
Ligations
Ligation 1: RBS and Vector
Ligation 2: no RBS and Vector
Materials for Ligation 1:
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of RBS insert |
4 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
9.5 |
Materials for Ligation 2
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of no RBS insert |
3 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
10.5 |
Transformation:
50 mL comp cells + 5 uL plasmid (shipment plasmid + RBS/no RBS)
On ice for 2 minutes
Heat shock 42 degree Celsius for 45 seconds
Ice 2 minutes
550 mL SOC media into mixture
Shake at 37 degree Celsius for 1 hour (recovery)
Plate onto CAM plate + incubate at 4 pm
Plan + CAM plate
Friday, July 18, 2014
Plan of Attack with pBRES36a
1. Digest with individual restriction enzymes and a negative control
a. No enzyme
b. XbaI
c. PstI
d. SpeI
e. EcoRI
2. Visualize on Gel
3. Double Digest with Pair from Step 1
4. Visualize on gel
5. Map the sequence
CAM plates made with 35 ug/mL
Transformation with:
1. Ligation product (2 of them)
2. Plasmid that's known to express CAM (for control)
7/20/14
2nd Double Digestion: For the purpose of confirming that there was no mix up in the previous digestion.
Components |
Amount (uL) |
10x Fast Digest Buffer |
2 |
pBRES36a (42ng/uL) |
16 |
SpeI |
1 |
PstI |
1 |
Total |
20 |
Lane # (from left) |
Sample |
1 |
Ladder (not visible) |
2 |
No enzyme |
3 |
PstI and SpeI |
4 |
No enzyme |
5 |
PstI and SpeI |
Housekeeping
Wednesday, July 23, 2014
Cam plates: 35 ug/mL, dilute 1:1000
Used 75 mL, used 75 ul
For 4 plates
Transformation with RBS:
50 mL E. coli comp cells, 5 uL RBS ligation
50 mL E. coli comp cells, 5 uL no RBS ligation
Transformed and recovered for 1 hour
Plated 50 uL of transformed cells onto cam plates
Restriction Digest of pBRES36a Plasmid
Monday, July 28, 2014
1. Digestion
Purpose: cut with restriction enzymes to provide a rough map for the pBRES36a plasmid and confirm the plasmid's identity
Materials:
Reagents |
Amount (ul) |
pBRES36a plasmid |
17 |
Buffer |
2 |
Restriction Enzyme |
1 |
Total Volume |
20 |
Procedures:
1. Mix reagents together and spin down gently.
2. Incubated for 10 minutes
3. Pipet up and down gently to mix samples
4. Load each sample onto the agarose gel
5. Run electrophoresis according to the conditions specified.
Enzyme Used:
1. XbaI
2. PstI
3. SpeI
4. EcoRI
2. Gel Electrophoresis
Run 1: Gel Set Up (7/18/14)
lane |
Reaction/Reagent |
Amount loaded (ul) |
1 |
1 kb Marker |
1 |
2 |
Plasmid + XbaI |
5 |
3 |
Plasmid + PstI |
5 |
4 |
Empty Lane |
0 |
5 |
Plasmid + SpeI |
5 |
6 |
Plasmid +EcoRI |
5 |
7 |
Plasmid only |
5 |
Run 1 Condition:
1. 2.0 hours
2. 80 Volts
3. 0.8% Agarose Gel
4. 1x TBE Buffer
Run 2: Gel Set Up (7/28/14)
lane |
Reaction/Reagent |
Amount loaded (ul) |
1 |
1 kb Marker |
1 |
2 |
Plasmid + XbaI |
5 |
3 |
Plasmid + PstI |
5 |
4 |
Plasmid + SpeI |
5 |
5 |
Plasmid +EcoRI |
5 |
6 |
Plasmid +SpeI and EcoRI |
5 |
7 |
Hsp60 + XbaI and PstI |
5 |
8 |
mCherry + XbaI and PstI |
5 |
Run 2 Condition:
1. 1.5 hours
2. 80 Volts
3. 0.8% Agarose Gel
4. 1x TBE Buffer
July 28, 2014 con’t:
Results
Run 1 (7/18/14) Left and Run 2 (7/28/14) Right
Ligation, Transformation, Selection
Thursday, July 24, 2014
Ligations
Ligation 1: RBS and Vector
Ligation 2: no RBS and Vector
Materials for Ligation 1:
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of RBS insert |
4 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
9.5 |
Materials for Ligation 2
Reagent |
Amount (ul) |
Ligase |
1 |
Buffer T4 |
2 |
30 ng of no RBS insert |
3 |
60 ng of vector plasmid |
3.5 |
ddH2O (Volume to 20) |
10.5 |
Incubate at RT for 15 minutes
Transformation
1. 50 mL E. coli comp cells
2. 6 uL plasmid
3. 2 min on ice
4. 45s heat shock at 42 degrees Celsius
5. 2 min on ice
6. 950 mL SOC growth media
7. 1 hour recovery at 37 degrees Celsius
Selection:
plate 60 mL of transformed cells onto selection plate (LB + CAM plates)
8/1/14
1. Take out overnight liquid culture around 11:00 am
2. Mini prep iGEM plasmid parts
Part |
Amount (ng/uL) |
Desaturase |
42.6 |
Cathelicidin |
12.7 |
mCherry |
12.7 |
8/4/14 Lab
Make 1% Gel (30 mL):
0.3 g Agarose
30 mL 1x TBE
1 uL EtBr
Restriction Digests:
hsp60 PCR products:
RBS: no RBS:
6 uL ddH2O 6 uL ddH2O
2 uL Buffer 2 uL Buffer
1 uL XbaI 1 uL XbaI
1 uL PstI 1 uL PstI
10 uL PCR product 10 uL PCR product
iGEM Parts:
mCherry: Cathelicidin: deSaturase:
6 uL ddH2O 6 uL ddH2O 6 uL ddH2O
2 uL FD Green Buffer 2 uL FD Green Buffer 2 uL FD Green Buffer
1 uL XbaI 1 uL XbaI 1 uL XbaI
1 uL PstI 1 uL PstI 1 uL PstI
10 uL mCherry 10 uL Cathelicidin 10 uL deSaturase
PCR Purification:
Run PCR purification of hsp60 digests (RBS and no RBS)
1: RBS concentration-
2: no RBS concentration –
Gel Electrophoresis:
Ladder – mcherry – cath – desat - empty - cath – mcherry – desat
Failed. Will upload pic later
Ligation:
Ligate hsp60 RBS and no RBS into pSB1C3 backbones
RBS: no RBS:
9.5 uL ddH2O 10.5 uL ddH2O
3.5 uL backbone 3.5 uL backbone
4 uL insert 3 uL insert
2 uL buffer 2 uL buffer
1 uL ligase 1 uL ligase
Transformation:
Transform RBS and no RBS ligations into E. coli competent cells
5 uL plasmid
100 uL competent cells
Ice 2 minutes
Heat shock (42C) 45s
Ice 2 minutes
Add 950 uL outgrowth media
Recover with shaking (37C) for 1 hour
Plate onto selection plate (chloramphenicol)
Restriction Digest:
Cut mCherry prep, Cath. Prep., deSat. Prep, 1 RBS, 1 no RBS, 1 mCherry with XbaI and PstI
6 uL ddH2O
2 uL FD Green Buffer
1 uL XbaI
1 uL PstI
10 uL plasmid DNA
Gel Electrophoresis:
1 kb+ ladder – mCherry prep – Cath. Prep – deSat prep – 1 RBS – 1 no RBS – 1 mCherry -
8/5/15 Lab
Make 1% Gel (35 mL)
0.35 g Agarose
35 mL 1x TBE
1 uL EtBR
Restriction Digests:
Master Mix (for 6 reactions): x6.5 =
6 uL ddH2O 39 uL
2 uL Green Buffer 13 uL
1 uL XbaI 6.5 uL
1 uL PstI 6.5 uL
Combine 10 uL of Master Mix with 10 uL of each plasmid:
mCherry Prep, Cath. Prep, deSat prep., 1 RBS, 1 no RBS, 1 mCherry
Incubate 45 mins at 37C
Gel Electrophoresis:
Run 1 hour 30 mins
Lanes:
1 kb+ ladder – mCherry mini – Desat – Cath – 1 RBS – 1 no RBS – 1 mCherry – 1 kb+ ladder
08/08/14
Used the culture collected from 8/1/14 mini-prep for the subsequent restriction digest
1. Restriction digest (45 minutes)
2. Run agarose gel
Plasmid |
Expected Band (kb) |
|
1 |
Desaturase |
~1 |
2 |
||
3 |
Cathelicidin |
~0.1 |
4 |
||
5 |
RBS |
~0.3 |
6 |
||
7 |
No RBS |
~0.3 |
8 |
Digestion was incomplete and need to repeat experiment
Experiment was repeated on 8/9/14 and digestion was successful. The desire bands were excised and purified. Unfortunately the image file was lost due to a technical problem prior to routine file backup. However, further experiments continued to use parts isolated on this date and bands of correct size were present through all of these steps.
Lab Work 8/12/14:
Gel Purification:
Gel Fragment |
Gel + Tube Mass (g) |
Tube Mass (g) |
Gel Mass (g) |
Membrane Binding Solution Added (uL) |
DNA Concentration (ng/uL) |
1 Cath. Part |
1.133 |
1.050 |
0.083 |
83 |
2.9 |
2 Cath. Part |
1.146 |
1.050 |
0.096 |
96 |
3.9 |
3 Desat. Part |
1.173 |
1.050 |
0.123 |
123 |
3.1 |
4 Desat. Part |
1.192 |
1.050 |
0.142 |
142 |
3.1 |
1 Backbone |
1.160 |
1.050 |
0.110 |
110 |
3.2 |
2 Backbone |
1.152 |
1.050 |
0.102 |
102 |
3.2 |
3 Backbone |
1.151 |
1.050 |
0.101 |
101 |
3.2 |
4 Backbone |
1.200 |
1.050 |
0.150 |
150 |
4.3 |
5 Backbone |
1.201 |
1.050 |
0.151 |
151 |
6.6 |
|
|
|
|
|
N/A |
7 Backbone |
1.243 |
1.050 |
0.193 |
193 |
5.7 |
8 Backbone |
1.159 |
1.050 |
0.109 |
109 |
5.7 |
Backbone |
1.300 |
1.050 |
0.250 |
250 |
4.4 |
*Add 10 uL of Membrane Binding Solution per 10 mg of Gel slice
-Vortex Gel and solution
-Incubate at 55C for 10 mins to melt gel
-Spin Down
-Gel Purify
Miniprep:
Miniprepped mRFP1 and MelA liquid cultures
-used same elution buffer for two of the same sample to obtain double DNA concentration
Concentrations:
1 mRFP: 516 ng/uL
2 mRFP: 462.5 ng/uL
3 mRFP: 243.5 ng/uL
1 MelA: 117.9 ng/uL
2 MelA: 158.3 ng/uL
3 MelA: 146.1 ng/uL
Lab Work 8/13/14:
Restriction Digest:
Digesting 1 mRFP, 2 mRFP, 3 mRFP, 1 MelA, 2 MelA, 3 MelA with XbaI and PstI.
20 uL Reactions: Master Mix:
5 uL Plasmid DNA 13 uL FD Green Buffer
2 uL FD Green Buffer 6.5 uL XbaI
1 uL XbaI 6.5 uL PstI
1 uL PstI 78 uL ddH2O
12 uL ddH2O 104 uL Total
*Reactions are 5 uL Plasmid + 15 uL Master Mix
*There are 6 reactions. Master Mix is 6.5x
*Incubate for 45 minutes at 37C
Gel Electrophoresis:
Running a gel to check the validity of the mRFP1 and MelA mini-prepped DNA.
Expect to see mRFP1 band at 706 bp and MelA band at 1844 bp.
Lanes:
1 kb+ --- 1 mRFP --- 2 mRFP --- 3 mRFP --- 1 MelA --- 2 MelA --- 3 MelA --- EMPTY
Gel:
Gel Purification:
Gel Fragment |
Gel + Tube Mass (g) |
Tube Mass (g) |
Gel Mass (g) |
Membrane Binding Solution Added (uL) |
DNA Concentration (ng/uL) |
1 mRFP |
1.142 |
1.017 |
0.125 |
||
2 mRFP |
1.143 |
1.009 |
0.134 |
||
3 mRFP |
1.149 |
1.012 |
0.137 |
*Did not complete Gel Purification
Plan for Tomorrow:
1) Finish Gel Purification (Possibly Re-Run. I believe I cut wrong fragment)
2) Make chloramphenicol plates
3) Resuspend YF1 & FixJ, Blue Light Sensor (Plate 1, Well 10N; 1:10N)
4) Resuspend FixK2 Promoter (Plate 1, Well 19G; 1:19G)
5) Transform and Plate (YF1 & FixJ) and FixK2 Promoter
If able to obtain Kanamycin:
1) Make Kanamycin plates (2-3)
2) Transform and Plate mCherry Bomb (3.4 uL of plasmid left)
Lab Work 8/14/14:
Plan:
1) Re-Run mRFP1 on Gel and then Gel Purify
2) Make Chloramphenicol Plates
3) Dilute mCherry Bomb and nanodrop
3a) If enough DNA is present, run PCR (1.4 ng/uL only)
3b) If not enough DNA is present, make Kanamycin plates
4) Resuspend YF1 & FixJ, Blue Light Sensor (Plate 1, Well 10N; 1:10N)
5) Resuspend FixK2 Promoter (Plate 1, Well 19G; 1:19G)
6) Transform and Plate (YF1 & FixJ) and FixK2 Promoter (Chloramphenicol)
7) Transform and Plate mCherry Bomb plasmid (Kanamycin)
mRFP1 Gel Electrophoresis:
-Create 25 mL or 1% agarose gel and let solidify
-Load digested mRFP1 DNA from 8/13/14
Lanes:
1 kb+ Ladder --- Empty --- 1 mRFP --- empty --- 2 mRFP --- empty --- 3 mRFP --- empty
Dilute mCherry Bomb and nanodrop:
-1 uL mCherry Bomb into 9 uL ddH2O
-Concentration of 1.4 ng/uL
Make Plates:
*1 Kanamycin; 3 Chloramphenicol
-Kanamycin working concentration of 50 ng/mL
-CAM working concentration of 25 ng/mL
Resuspend DNA:
*Added 10 uL ddH2O to each part to resuspend
Transform:
Transformed Blue Light Sensor, Blue Light Promoter, and mCherry Bomb
*Plates put in 37C incubator at 4:16 PM
Gel Purification:
Gel Fragment |
Gel + Tube Mass (g) |
Tube Mass (g) |
Gel Mass (g) |
Membrane Binding Solution Added (uL) |
DNA Concentration (ng/uL) |
1 mRFP |
1.195 |
1.014 |
0.181 |
181 |
3.6 |
2 mRFP |
1.217 |
1.002 |
0.215 |
215 |
2.0 |
3 mRFP |
1.158 |
1.012 |
0.146 |
146 |
4.6 |
Lab Work 8/15/15:
Morning:
Took out agar plates of Blue Light Sensor, Blue Light Promoter, and mCherry Bomb
*Colonies were spotted on each Plate
Evening:
Made liquid cultures for Blue Light Sensor, Blue Light Promoter, and mCherry Bomb
*5 mL per liquid culture
*2 Liquid cultures per plasmid
*Added CAM to Blue Light Sensor and Blue Light Promoter cultures at working concentration of 25 ng/mL
*Added Kan to mCherry Bomb culture at working concentration of 50 ng/mL
Lab Work 8/16/14:
Miniprep:
-Miniprepped liquid cultures of Blue Light Promoter, Blue Light Sensor, and mCherry Bomb
Concentrations:
Blue Light Promoter (1:19G): 44.5 ng/uL
Blue Light Sensor (1:10N): 36.6 ng/uL
mCherry Bomb: 35 ng/uL
Lab Work 8/18/14:
PCR:
-PCRing hsp60 out of plasmid mCherry Bomb
Master Mix |
Primers and DNA |
PCR Reaction |
|||||
Reagent |
1x Rxn (uL) |
2.5x Rxn (uL) |
Primer |
RBS (uL) |
no RBS (uL) |
Reagent/DNA |
Volume (uL) |
Buffer |
10 |
25 |
Fwd Primer |
2.5 |
2.5 |
Master Mix |
11.5 |
dNTP |
1 |
2.5 |
Rev Primer |
2.5 |
2.5 |
Primers and DNA |
7.5 |
Phusion |
0.5 |
1.25 |
mCherry Bomb |
2.5 |
2.5 |
ddH2O |
31 |
Total |
11.5 |
28.75 |
Total |
7.5 |
7.5 |
Total |
50 |
*Two 20 uL PCR reactions of each (RBS and no RBS) were carried out
PCR conditions:
98C for 30s
98C for 10s
66C for 30s x30 cycles
72C for 30s
72C for 5 min
4C holding
Gel Electrophoresis:
Ran two PCR products with a ladder on a gel. There were no bands shown. PCR was unsuccessful
Lab Work 8/19/14:
Restriction Digest:
Digesting Blue Light Sensor and Blue Light Promoter
20 uL Reactions:
5 uL Plasmid DNA
2 uL FD Green Buffer
1 uL XbaI
1 uL PstI
12 uL ddH2O
*Incubate for 45 minutes at 37C
Gel Electrophoresis:
Running a gel to check the validity of the Blue Light Sensor (1796 bp), Blue Light Promoter (250 bp), and PCR products (~380 bp).
Lanes:
1 kb+ ladder --- RBS PCR --- no RBS PCR --- Blue Light Sensor --- Blue Light Sensor --- Blue Light Promoter --- Blue Light Promoter --- empty
9/12/14
Goal:
1. PCR purify Hsp60 with and without RBS
2. Digestion of purified product with XbaI and PstI
3. Gel purification of digestion products
a. Cut out slices and store at 4 ℃
Experiments:
Tubes Labeled by MJ
Tube label |
Content |
1, PCR purification product, no RBS |
1st elution: PCR purification product, no RBS |
2, PCR purification product, no RBS |
2nd elution: PCR purification product, no RBS |
1, PCR purification product, RBS |
1st elution: PCR purification product, RBS |
2, PCR purification product, RBS |
2nd elution: PCR purification product, RBS |
1. PCR purification
Step |
Hsp60 with RBS (uL) |
Components |
Hsp60 without RBS (uL) |
1 |
34.5 |
Membrane binding solution |
34.5 |
2 |
700 |
Membrane Wash solution |
700 |
3 |
500 |
Membrane Wash solution |
500 |
4 |
50 |
1st elution with Nuclease free ddH2O |
50 |
5 |
30 |
2nd elution with Nuclease free ddH2O |
30 |
2. Digestion of purified product
Components |
Amount for Hsp60 with RBS (uL) |
Amount for Hsp60 without RBS (uL) |
10x Fast Digest Green Buffer |
2 |
2 |
PCR Product of Hsp60 |
7 |
7 |
XbaI |
1 |
1 |
PstI |
1 |
1 |
ddH2O |
13 |
13 |
Total |
24 |
24 |
Incubated at 37 ℃ for 30 minutes
2 reactions each were digested for 1st elution of both with and without RBS; 1 reaction each for the 2nd elution for both with and without RBS
3. Gel Set Up:
Lane |
Sample |
Amount (uL) |
Expected band size (bp) |
1 |
1kb DNA Ladder |
2 |
None |
2 |
Elution 1, no RBS digested |
5 |
~ 300 bp |
3 |
Elution 1, no RBS digested |
5 |
|
4 |
Elution 2, no RBS digested |
5 |
|
5 |
1kb DNA Ladder |
2 |
None |
6 |
Elution 1, RBS digested |
5 |
~300 bp |
7 |
Elution 1, RBS digested |
5 |
|
8 |
Elution 2, RBS digested |
5 |
4. Mass of tube and tube plus sample
Tube label |
Sample (lane #) |
Mass of tube (g) |
Mass of Tube plus sample (g) |
1 |
2 |
1.012 |
1.1 |
2 |
3 |
1.005 |
1.098 |
3 |
4 |
1.018 |
1.121 |
4 |
6 |
1.01 |
1.105 |
5 |
7 |
1.02 |
1.113 |
6 |
8 |
1.003 |
1.094 |
Goals:
1) Digestion of Blue Light Parts w/ X+P
2) Run Parts on Gel
3) Gel Purify Blue Light Parts, hsp60 parts, vector backbone
4) Ligate hsp60 + backbone
Digestion:
9 uL ddH2O
2 uL Fast Digest Green Buffer
1 uL XbaI
1 uL PstI
7 uL Plasmid (Blue Light Sensor and Blue Light Promoter)
20 uL rxn
Incubate at 37 C for 30 min
Gel Electrophoresis:
Lanes:
1 kb+ ladder – Blue Sensor – Blue Sensor – Blue Promoter – Blue Promoter – empty
Image at 30 mins: Image at 1 hour, 15 mins:
*Lanes 4/5 extracted at 30 mins
Purification:
Tube Weight (g) |
Tube + Gel Weight (g) |
Gel Fragment Weight (g) |
Membrane Binding Solution (uL) |
|
Backbone |
1.021 |
1.183 |
0.162 |
162 |
Blue Promoter |
1.016 |
1.198 |
0.182 |
182 |
Backbone |
1.016 |
1.187 |
0.171 |
171 |
Blue Sensor |
1.004 |
1.126 |
0.122 |
122 |
no RBS |
0.088 |
88 |
||
no RBS |
0.093 |
93 |
||
RBS |
0.095 |
95 |
||
RBS |
0.093 |
93 |
Ligation:
RBS no RBS
1 uL ligase 1 uL ligase
2 uL Buffer 2 uL Buffer
4 uL Insert 3 uL Insert
3.5 uL Backbone 3.5 uL Backbone
9.5 uL ddH2O 10.5 uL ddH2O
20 uL 20 uL
9/22/14
Goals:
1. Digestion of desaturase, and mRFP-1
2. Purification of desaturase and mRFP-1
3. Ligation into the vector
4. Digestion of 7 uL of plasmid DNA
5. Purification
Experiment:
Cut mRPF-1 with EcoRI and XbaI
Desaturase with EcoRI and SpeI
Components mRFP-1 |
Amount (uL) |
Components desaturase |
10x Fast Digest Buffer |
2 |
10x Fast Digest Buffer |
plasmid |
7 |
plasmid |
EcoRI |
1 |
EcoRI |
XbaI |
1 |
SpeI |
ddH2O |
9 |
ddH2O |
Total |
19 |
Total |
Gel:
Lane |
1 |
2 |
3 |
4 |
Components |
1kb DNA Ladder |
Desaturase (EcoRI+SpeI) |
||
Amount (uL) |
2 |
5 |
5 |
5 |
Lane |
5 |
6 |
7 |
8 |
Components |
1kb DNA Ladder |
mRFP-1 (EcoRI+XbaI) |
||
Amount (uL) |
2 |
5 |
5 |
5 |
9/24/14
mRFP-1 and desaturase plasmids were previously collected and this is the resulting digestion (9/22/14) followed by gel purification of the previous experiments.
1. Gel purification: and resulting concentrations:
Tube label |
Part excised |
Mass (g) |
Concentration (ng/uL) |
1 |
Desaturase Vector Backbone |
Not used since this is the backbone |
|
2 |
|||
3 |
|||
4 |
Desaturase Part |
0.124 |
3.9 |
5 |
0.123 |
4.7 |
|
6 |
0.095 |
4.2 |
|
7 |
mRFP-1 linearized plasmid |
0.095 |
4.4 |
8 |
0.103 |
4.4 |
|
9 |
0.133 |
4.8 |
9/26/14
1. Ligation reaction BH
Components |
Amount (uL) |
Insert (desaturase) |
9 |
Vector Backbone (single cut mRFP-1) |
3.5 |
Ligase |
1 |
Buffer |
1.5 |
ddH2O |
5 |
Total |
20 |
Negative control: just VB (labeled A)
Ligation 1 (labeled B)
Ligation 2 (labeled C)
2. Transformation into E. coli competent cells BH
Incubation at 37 ℃ on LB + CAM from 2 pm
Note: the plate was examined once at 6 hours and another time at 8 hours— yielded no usable colonies
3. Mini-Prep (BH) of colonies culture grown by MJ
Labeling maintained the same (1-6)
Resuspended in 50 uL of elution buffer
Undetermined concentration
Ligation and Transformation were repeated 2 times yet the reaction never yielded colonies that survived in LB+CAM culture.
Lab Work 9/29/14:
Diagnostic Digest:
Hsp60(no RBS) – mRFP1 (~1090 bp) 1) X+P
Blue Promoter – mRFP1 (~1050 bp) 2) X+P, 3) S+P
Hsp60 (no RBS) – Blue Sensor (~2160 bp) 4) X+P
MelA (~1896 bp) 5) X+P
20 uL Rxns
9 uL ddH20
2 uL FD Green
1 uL XbaI/SpeI
1 uL PstI
7 uL Plasmid
*Incubate for 30 min @ 37C
Gel Electrophoresis:
1 kb+ ladder – 1 – 2 – 3 – 5 – 4 – empty
Gel @ 30 mins Gel @ 1 hr, 30 mins
*Note: Band 3 was extracted after 30 minutes. The lower of band 5 was extracted at 1 hr, 30 min
Gel Extraction:
Blue Promoter – mRFP1 S+P gel weight: 0.086 g
Hsp60 (no RBS) – Blue Sensor X+P gel weight: 0.062 g
Made 6 CAM plates & 2 AMP plates
Transformation:
Transformed RBS (4:1N) and Terminator (4:16G).
Plate onto AMP plates
Lab Work 10/1/14:
Mini Prep:
Mini Prep RBS and Terminator Liquid Cultures
Gel Purify:
Add 86 uL Membrane Binding Solution to Blue Promoter – mRFP1 (S+P) part
Add 62 uL Membrane Binding Solution to hsp60 (no RBS) – Blue Sensor (X+P) part
Restriction Digests:
Terminator (E+X) (~2150 bp) Cathelicidin (E+S) (~123 bp)
RBS (E+X) (~2082 bp) Blue Promoter (E+S) (~250 bp)
20 uL rxns:
9 uL ddH2O
2 uL FD Green
1 uL EcoRI
1 uL XbaI/SpeI
7 uL Plasmid
Incubate 30 mins @ 37C
Gel Electrophoresis:
Lanes:
1 kb+ ladder – Terminator (E+X) – Cathelicidin (E+S) – RBS (E+X) – B. Promoter (E+S) – empty
*Run Gel 15 minutes
Gel Purifiy:
RBS (E+X)
Blue Promoter (E+S)
Gel Fragment |
Gel + Tube Mass (g) |
Tube mass (g) |
Gel Mass (g) |
Membrane Binding Solution (uL) |
DNA Concentration (ng/uL) |
|
|
|
|
|
|
|
|
|
|
|
|
RBS (E+X) |
1.033 |
0.985 |
0.048 |
48 |
1.7 |
Blue Promoter (E+S) |
1.082 |
1.005 |
0.077 |
77 |
1.8 |
Ligation:
Blue Promoter – RBS
15 uL Rxn:
3.5 uL Insert (Blue Promoter)
9 uL VB (RBS)
1.5 uL Buffer
1 uL Ligase
0 uL ddH2O
Transformation:
Transform Blue Promoter – RBS onto AMP plate
Lab Work 10/4/14:
Morning:
Made Liquid Cultures of Blue Promoter – RBS part in Turbo E.coli Comp Cells
Afternoon:
Mini Prep:
Mini Prepped Blue Promoter – RBS : ??? ng/uL
Restriction Digest:
Blue Promoter – RBS (X/P)
Blue Promoter – RBS (S/P)
Terminator (S/P)
20 uL rxns:
9 uL ddH2O
2 uL FD Green
1 uL XbaI/SpeI
1 uL PstI
7 uL Plasmid
Gel Electrophoresis:
Lanes:
1) 1 kb+ ladder
2) Blue Promoter – RBS (X+P) (~262 bp)
3) Blue Promoter – RBS (S+P) (~2350 bp)
4) Terminator (S+P) (~2150 bp)
Gel Purify:
Gel Fragment |
Tube (g) |
Gel + Tube (g) |
Gel (g) |
Membrane Binding Solution (uL) |
Concentration (ng/uL) |
Blue Promoter-RBS (S/P) |
1.002 |
1.067 |
0.065 |
65 |
5.0 |
Terminator (S/P) |
1.020 |
1.093 |
0.073 |
73 |
2.1 |
Ligation:
1) Blue Promoter – mRFP1 (S/P) (~3000 bp) + Cathelicidin (X/P) (~120 bp)
2) Blue Promoter – RBS (S/P) (~2350 bp) + Cathelicidin (X/P) (~120 bp)
3) Terminator (S/P) (~2150 bp) + hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp)
4) Blue Promoter – mRFP1 (S/P) (~3000 bp) + hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp)
Ligation |
VB bp |
VB concentration (ng/uL) |
Insert bp |
Insert concentration (ng/uL) |
VB (uL) |
Insert (uL) |
1 |
3000 |
2.9 |
120 |
3.9 |
4.00 |
8.50 |
2 |
2350 |
5.0 |
120 |
3.9 |
3.50 |
9.00 |
3 |
2150 |
2.1 |
2160 |
4.9 |
3.00 |
9.50 |
4 |
3000 |
2.9 |
2160 |
4.9 |
3.50 |
9.00 |
1.5 uL Buffer
1 uL Ligase
Transformation:
Blue Promoter – mRFP1 – Cathelicidin (CAM)
Blue Promoter – RBS – Cathelicidin (AMP)
Terminator – hsp60 (no RBS) – Blue Sensor (AMP)
Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (CAM)
Lab Work 10/5/14:
Ligation:
Ligation 3: Terminator (S/P) + hsp60 (no RBS) – Blue Sensor (X/P)
1.5 uL Buffer
1 uL Ligase
6 uL VB (Terminator)
6.5 uL Insert (hsp60-Blue Sensor)
Transformation:
Transform 3: Terminator – hsp60 (no RBS) – Blue Sensor
AMP Plate
MiniPrep:
Mini 1: Blue Promoter – mRFP1 – Cathelicidin 61.5 ng/uL
Mini 2: Blue Promoter – RBS – Cathelicidin 85.9 ng/uL
Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor 81.0 ng/uL
Restriction Digest:
1) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (X/P)
2) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (S/P)
3) Mini 2: Blue Promoter – RBS – Cathelicidin (X/P)
4) Mini 2: Blue Promoter – RBS – Cathelicidin (S/P)
5) Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)
20 uL rxns:
9 uL ddH2O
2 uL FD Green
1 uL XbaI/SpeI
1 uL PstI
7 uL Plasmid
Gel Electrophoresis:
Lanes:
1) 1 kb+ ladder
2) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (X/P) (~1000 bp) (Part)
3) Mini 1: Blue Promoter – mRFP1 – Cathelicidin (S/P) (~3100 bp) (VB)
4) Mini 2: Blue Promoter – RBS – Cathelicidin (X/P) (~370 bp) (Part)
5) Mini 2: Blue Promoter – RBS – Cathelicidin (S/P) (~2470 bp) (VB)
6) Mini 4: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)(~3160 bp) (Part)
*After 30 mins, hsp60 (no RBS) – Blue Sensor (X/P) (~2160 bp) added to lane 7. Ladder added lane 8.
Gel Purify:
Gel Fragment |
Tube (g) |
Gel + Tube (g) |
Gel (g) |
Membrane Binding Solution (uL) |
Concentration (ng/uL) |
Mini 1: B.P. - mRFP1 - Cath (S/P) |
1.013 |
1.072 |
0.059 |
59 |
2.6 |
no RBS - Blue Sensor (X/P) |
1.016 |
1.065 |
0.049 |
49 |
1.2 |
Ligation:
1.5 uL Ligation Buffer
1 uL Ligase
Lig 1: Lig 2:
9 uL Blue Promoter (Insert) (~200 bp) 6 uL Terminator (VB) (~2100 bp)
3.5 uL RBS (VB) (~2100 bp) 6.5 uL hsp60 (no RBS) – B. Sensor (Insert) (~2160)
Lig 3:
3.5 uL B. Prom – mRFP1 (VB) (~3100)
9 uL hsp60 (no RBS) – B. Sensor (Insert) (~2160 bp)
Transformation:
Transform 1: Blue Promoter – RBS onto AMP plate
Transform 2: Terminator – hsp60 (no RBS) – Blue Sensor onto AMP plate
Transform 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor onto CAM plate
Make Liquid Cultures:
Made 2 5mL Liquid cultures of LB + AMP, Blue Promoter – RBS
Lab Work 10/6/14:
Make Liquid Cultures:
(AMP) Transform 1: Blue Promoter – RBS
(AMP) Transform 2: Terminator – hsp60 (no RBS) – Blue Sensor
(CAM) Transform 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor
Mini prep:
Mini 1: Blue Promoter – RBS
Mini 2: Terminator – hsp60 (no RBS) – Blue Sensor
Mini 3: Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor
Restriction Digest:
1) Blue Promoter – RBS (X/P)
2) Blue Promoter – RBS (S/P)
3) Terminator – hsp60 (no RBS) – Blue Sensor (X/P)
4) Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P)
20 uL Rxns
9 uL ddH2O
2 uL FD Green
1 uL XbaI/SpeI
1 uL PstI
7 uL Plasmid
Gel Electrophoresis:
Lanes:
1) 1 kb+ ladder
2) Blue Promoter – RBS (X/P) (~262 bp)
3) Blue Promoter – RBS (S/P) (~2341)
4) Terminator – hsp60 (no RBS) – Blue Sensor (X/P) (~2236)
5) Blue Promoter – mRFP1 – hsp60 (no RBS) – Blue Sensor (X/P) (~3100)
*Note: In lane four, the VB is ~2100, will need long separation to see. Part will be bigger than backbone
*Stop at 15 mins and check lane 2, if correct band is present, extract lane 3 before continuing.
*Run for another hour and fifteen before stopping a second time and extracting lane 4
Gel Purify:
Ligation:
Ligation 1: Blue Promoter – RBS (X/P) + Cathelicidin (S/P)
Ligation 2: Blue Promoter – mRFP1 – Cathelicidin (S/P) + Terminator – hsp60 (no RBS) – Blue Sensor (X/P)
Ligation |
VB bp |
VB Concentration (ng/uL) |
Insert bp |
Insert Concentration (ng/uL) |
VB (uL) |
Insert (uL) |
|
|
|
|
|
|
|
2 |
2500 |
2236 |