Team:Paris Saclay/Notebook/October/6
Contents
Thursday 6th October
Lab work
Visualization
Colony PCR of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)
By Maxence
For that purpose, X 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 | 1 min | |
| Final Extension | 72°C | 7 min |
| Hold | 4°C | $\infty$ |
Primers used were:
| Matrix | Clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) |
|---|---|
| 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 - FKBP - GFP 10 | 1714 |
GEL
