Team:Paris Saclay/Notebook/August/25
Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB
Mahnaz
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) were made from these clones. Both petri dishes and liquid cultures were grown at 37°C.
For each clones contained in 20 μl water, 4.13 μL of the following mix were added :
- 2.5 µL DreamTaq Buffer
- 0.5 µL of dNTPs (10mM)
- 0.5 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 | 94°C | 30 sec |
| Tm | 30 sec | |
| 72°C | t | |
| Final Extension | 72°C | 7min |
| Hold | 4°C | $\infinity\$ |
Primers used were:
| Matrix | plasmid pJET coding sg-Nm | plasmid pJET coding FRB |
|---|---|---|
| Primers | pJET R and pJET F | |
| Tm | 60.0C | |
| t | 30 sec |
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 NM - GFP 10 - pSB1C3 | 3688 |
GEL GEL GEL
