Thuersday 22nd September
Lab work
Visualization
Colony PCR of 16 clones containing FRB - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_022)
By Maxence, Mahnaz & Coline
A colony PCR was done for 16 clones from the 20th September. For that purpose, 16 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
|
1500
|
GEL
Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_019)
By Mahnaz
As sequencing results were not convincing, a new colony PCR was done for 16 clones from the 15th September but with primers iPS168 & iPS169. 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, 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
|
$\infinity\$
|
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
|
