Thursday 22nd September
Lab work
Visualization
Colony PCR of 8 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
By Maxence, Mahnaz & Coline
A colony PCR was done for 8 clones (4 clones with GFP 1.9 PCR product obtained with DMSO and 4 clones with GFP 1.9 PCR product obtained without DMSO) from the 20th September. 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
|
1min 30sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
|
| 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)
|
| FRB - GFP 11 - GFP 1.9
|
1800
|
PCR products were not at the good size.
Colony PCR of 12 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)
By Maxence, Mahnaz & Coline
As sequencing results were not convincing, a new colony PCR was done for 12 clones from the 15th September (8 new clones and the 4 sent for sequencing) but with primers iPS168 & iPS169. For that purpose, 12 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
|
30 sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
|
| Primers
|
iPS83 and iPS84
|
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)
|
| FKBP - GFP 10 in pSB1C3 (pPS16_019)
|
757
|
PCR products were not at the good size, plasmids were empty.
PCR on cleaned up PCR products FKBP from 8th September and gblock 2.2 from the 8th September in order to fuse fuse these fragments
By Maxence, Manhaz & Coline
As sequencing and colony PCR of clones containing FKBP - GFP 10 in pSB1C3 did not show good results, a new clonage strategy was performed: before the Gibson, the two inserts were fused together by PCR. So PCR was performed with cleaned up PCR products FKBP (obtained the 8th September) and claned up PCR product gblock 2.2 (obtained the 8th September) with the following protocol.
For each 50μl of reaction, mix the following reagents :
- 1 µL of each PCR product
- 1 µL of dNTPs (10mM)
- 2 µL of each primer mix (10µM)
- 10 µL of buffer (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
|
| 65°C
|
30sec
|
| 72°C
|
20sec
|
| Final Extension
|
72°C
|
7min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
PCR products FKBP from 8th September and gblock 2.2 from the 8th September
|
| Primers
|
iPS145 and iPS84
|
Gel of PCR products
By Maxence, Mahnaz & Coline
After amplification, the totality of 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)
|
| FKBP - GFP 10
|
747
|
PCR product was at the good size.
Clean-up of PCR products from gel
By Maxence, Mahnaz & Coline
The PCR products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol. Their concentrations were assessed by NanoDrop:
Colony PCR of 8 clones containing corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017)
By Maxence, Mahnaz & Coline
A colony PCR was done for 8 clones from the 20th September. 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
|
| 25 cycles
|
95°C
|
30 sec
|
| 48.4°C
|
30 sec
|
| 72°C
|
4min
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
|
| Primers
|
iPS168 and iPS169
|
Colony PCR of 8 clones containing GFP 1.9 in pSB1C3 (pPS16_020)
By Maxence, Mahnaz & Coline
As sequencing results were not good, a new colony PCR was done for 8 clones from the 12th September. 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
|
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
|
Gel of PCR products
By Maxence, Mahnaz & Coline
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)
|
| dCas9 ST - GFP 11
|
3800
|
| GFP 1.9 in pSB1C3
|
1135
|
For GFP 1.9 in pSB1C3, all PCR products were at the good size, clones 3, 4, 7 and 8 were selected for sequencing. For dCas ST - GFP 11, 6 PCR products were at the good size, clones 2, 7 and 8 were selected for sequencing.
