Monday 19th September
Lab work
Visualization
Glycerol stocks of clones 7, 8, 9 and 10 containing FKBP - GFP 10 in pSB1C3 (pPS16_018)
"By Maxence, Mahnaz, Coline & Caroline"
The glycerol stock of the bacteria with the following plasmids were made.
- pPS16_018 (FKBP - GFP 10) clone 7
- pPS16_018 (FKBP - GFP 10) clone 8
- pPS16_018 (FKBP - GFP 10) clone 9
- pPS16_018 (FKBP - GFP 10) clone 10
500µL of liquid culture and 250 µL of glycerol 60% were mixed and put at -20°C.
Plasmids extraction of clones 7, 8, 9 and 10 containing GFP 1.9 in pSB1C3 (pPS16_018)
"By Maxence, Mahnaz Coline & Caroline"
The following plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- pPS16_018 (FKBP - GFP 10) clone 7
- pPS16_018 (FKBP - GFP 10) clone 8
- pPS16_018 (FKBP - GFP 10) clone 9
- pPS16_018 (FKBP - GFP 10) clone 10
Colony PCR of X clones containing GFP 1.9 in pSB1C3 (pPS16_020)
By Maxence, Mahnaz, Coline & Caroline
Seqencing of clones 2, 7, 8 and 12 from the 14th September was not good as empty plasmids were obtained. Another colony PCR was done for 16 clones from the 12th September. For that purpose, 16 were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 30 µg/mL Cm and liquid cultures (LB + 30 µg/mL Cm) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
For each clones contained in 20 μl water, 4.13 μL of the following mix were added :
- 2.5 µL DreamTaq Buffer
- 0.5 µL of dNTPs (10mM)
- 1 µL of each primer mix (10µM)
- 0.13 μl of DreamTaq Pol
PCR was performed as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
95°C
|
3 min
|
| 30 cycles
|
95°C
|
30 sec
|
| 72°C
|
30 sec
|
| 72°C
|
30sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Clones containing GFP 1.9 in pSB1C3 (pPS16_020)
|
| Primers
|
iPS84 and iPS140
|
After amplification, 3 µL of each PCR products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
| PCR products
|
Expected band size (bp)
|
| GFP 1.9 in pSB1C3
|
862
|
GEL good
All PCR products were at the good size. Clones 2, 7, 8 and 12 were selected and were grown at 37°C overnight.
PCR of pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 to correct mutations
By Maxence, Mahnaz, Coline & Caroline
In order to correct mutations in dCas9 ST - GFP 11, PCR was performed with the following protocol.
For each 50μl of reaction, mix the following reagents :
- 1 µL of matrix
- 1 µL of dNTPs (10mM)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer HF (5X)
- 0,5 µL of Phusion polymerase
- 32.5 µL of nuclease free water
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
Perform PCR as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
98°C
|
30sec
|
| 30 cycles
|
98°C
|
10sec
|
| 72°C
|
30sec
|
| 72°C
|
t
|
| Final Extension
|
72°C
|
5min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
dCas9 ST - GFP 11 (pPS16_017) clone 8
|
dCas9 ST - GFP 11 (pPS16_017) clone 8
|
| Primers
|
iPS174 and iPS175
|
iPS173 and iPS176
|
| Tm
|
72°C
|
72°C
|
| t
|
3min
|
50sec
|
Gel of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8
By Maxence, Mahnaz, Coline & Caroline
4 µL of each PCR products and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
PCR products expected were :
| PCR products
|
Expected band size (bp)
|
| Product 1 obtained by iPS174 & iPS175
|
5837
|
| Product 2 obtained by iPS173 & iPS176
|
1599
|
GEL
