Wednesday 5th October
Visualization
Colony PCR of 13 clones containing GFP 1.9 in pSB1C3 (pPS16_020)
By Maxence & Yacine
For that purpose, 13 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
|
30 sec
|
| 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
|
1135
|
No PCR products were obtained.
Plasmids extraction of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4 and FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6
By Maxence & Yacine
The following plasmids were extracted using a "standard Plasmid Miniprep":
- pPS16_018 (FKBP - GFP 10) clone 6
- pPS16_019 (FRB - GFP 11) clone 4
Cloning of FRB - GFP 11 (pPS16_019) in FKBP - GFP 10 - pSB1C3 (pPS16_018) by digestion-ligation
By Maxence & Yacine
For that purpose, FRB - GFP 11 (pPS16_019) clone 4 was cut by restriction enzymes XbaI & PstI as following:
- 20 µL of FRB - GFP 11 (pPS16_019) clone 4
- 4 µL of buffer FD
- 2 µL of restriction enzyme XbaI
- 2 µL of restriction enzyme PstI
- 10 µL of water
And FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6 was cut by restriction enzymes PstI & XbaI and was treated by the alkaline phosphatase FastAP (dephosphorylation of cloning vector DNA to prevent recircularization during ligation) as following:
- 20 µL of FKBP - GFP 10 - pSB1C3 (pPS16_018) clone 6
- 4 µL of buffer FD
- 2 µL of restriction enzyme SpeI
- 2 µL of restriction enzyme PstI
- 2 µL of alkaline phosphatase FastAP
- 10 µL of water
The mix were incubated for 20 minutes at 37°C. Then, 40 µL of each digestion products and 5 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
Migration products expected were :
| Migration products
|
Expected band size (bp)
|
| Template digested (pPS16_019 treated by XbaI & PstI)
|
727
|
| Vector digested (pPS16_018 treated by SpeI & PstI)
|
2784
|
The digestion products were cleaned up from the gel by using the NucleoSpin Gel and PCR Clean-up kit protocol.
