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 FKBP - GFP 10 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 8 clones containing GFP 1.9 in pSB1C3 (pPS16_020) from the 12th September
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 8 clones from the 12th September but anothers primers were used.
For that purpose, 8 clones 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, 5.13 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
|
| 48.4°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
|
iPS168 and iPS169
|
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
|
1135
|
Furthermore, plasmid extacted from clones containing GFP 1.9 in pSB1C3 (pPS16_018) were also put on the gel.
Plasmids extracted from clones containing GFP 1.9 in pSB1C3 (pPS16_018) were good, but no PCR products were obtained for GFP 1.9, cloning failed.
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
|
4837
|
| Product 2 obtained by iPS173 & iPS176
|
1599
|
PCR products were obtained at the good size.
PCR Clean-up of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8
By Maxence, Mahnaz, Coline & Caroline
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Linearization of cleaned-up PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8
By Maxence, Mahnaz, Coline & Caroline
Cleaned-up PCR product has been linearized for further Gibson application by using DpnI treatment :
- 30 µL of cleaned up PCR product
- 4 µL of fast digest buffer
- 1 µL of DpnI
- 5 µL of water
The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Clean-up of PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 treated by DpnI
By Maxence, Mahnaz, Coline & Caroline
PCR products treated by DpnI were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
