Wednesday 12th October
Visualization
Colony PCR of 12 clones containing GFP 1.9 ligated to PCR blunt
By Sylvie
For that purpose, 12 clones (6 with GFP 1.9 PCR product obtained with pUC19 and 6 with GFP 1.9 PCR product obtained by gblock) were screened and used for the usual protocol of Colony PCR.
For each clones contained in 2 μl liquid culture, 23 μL of the following mix were added :
- 2.5 µL DreamTaq Buffer
- 2 µL of dNTPs (10mM)
- 1 µL of each primer mix (10µM)
- 0.25 μl of DreamTaq Pol
- 17.25 μl of water
PCR was performed as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
95°C
|
5 min
|
| 30 cycles
|
95°C
|
30 sec
|
| 50°C
|
30 sec
|
| 72°C
|
30 sec
|
| Final Extension
|
72°C
|
10 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
|
862
|
Clones 3, 4, 5, 7, 8 & 9 were selected and plasmids were extracted using the "standard Plasmid Miniprep" protocol.
Extracted plasmids from clones 3, 4, 5, 7, 8 & 9 were digested by restriction enzyme NotI:
- 4 µL of plasmid
- 2 µL of buffer FD
- 2 µL of restriction enzyme NotI
- 12 µL of water
The mix were incubated for 30 minutes at 37°C. Then, 20 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
Cloning of GFP 1.9 in pSB1C3 by digestion-ligation
By Sylvie
Once template and vector were cut (14th October), DNA ligase was used to join the sticky ends of the template and vector together:
- 4.7 µL of template (GFP 1.9 PCR product treated by XbaI & PstI)
- 2 µL of vector (pSB1C3 treated by PstI & XbaI)
- 1.5 µL of Buffer T4 10X
- 1 µL of ligase T4 enzyme
- 5.8 µL of water
The mix was incubated for 30 minutes at rooming temperature.
Cloning of GFP 1.9 in FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) by digestion-ligation
By Sylvie
Once template and vector were cut (14th October), DNA ligase was used to join the sticky ends of the template and vector together:
- 3 µL of template (GFP 1.9 PCR product treated by XbaI & PstI)
- 7 µL of vector (pSB1C3 treated by PstI & SpeI)
- 1.5 µL of Buffer T4 10X
- 1 µL of ligase T4 enzyme
- 2.5 µL of water
Transformation of DH5a cells with GFP 1.9 in pSB1C3(pPS16_020) and FRB - FKBP - GFP 10 - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_023)
By Sylvie
Dh5a cells were transformed with GFP 1.9 in pSB1C3(pPS16_020) and FRB - FKBP - GFP 10 - GFP 11 - GFP 1.9 in pSB1C3 (pPS16_023) or controls (digested plasmid) using the usual protocol.
Co-transformation of DH5a cells with FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) + GFP 1.9 in pUC19 (pPS16_009) and FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) + GFP 1.9 ligated to PCR blunt
By Sylvie
Dh5a cells were co-transformed with pSB1C3 containing FRB - GFP 11 - FKBP - GFP 10 (pPS16_021) + pUC19 containing GFP 1.9 (pPS16_009) and FRB - GFP 11 - FKBP - GFP 10 (pPS16_021) + GFP 1.9 ligated to PCR blunt (clones 3, 4, 5, 7, 8 & 9) using the usual protocol. For that purpose, petri dishes used contained LB + Amp + Cm and two concentration of plasmids were used: 1 µL of plasmids not diluted and 1 µL of plasmids diluted 10X.
