Team:Paris Saclay/Notebook/August/29
PCR sur colonie
PCR of the plasmid extraction from clonies 4,5,9,12 containing FRB sequence in pJET plasmid
By Mahnaz
- 2.5 µL DreamTaq Buffer
- 0.5 µL of dNTPs (10mM)
- 1 µL of each primer (10µM) primer pJET R and F
- 0.13 μl of DreamTaq Pol
- 20 µl H2O
PCR was performed as follow:
| Step | Temperature | Time |
|---|---|---|
| Initial denaturation | 95°C | 3 min |
| 30 cycles | 95°C | 30 sec |
| 52 | 30 sec | |
| 72°C | 1 min | |
| Final Extension | 72°C | 7min |
| Hold | 4°C | \infinity\ |
Primers used were:
| Matrix | Clones containing dCas9 NM - GFP 10 in pSB1C3 |
|---|---|
| Primers | pJET R and F |
| Tm | 52°C |
| t | 1 min 30 |
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 in pJET | 474 |
NanoDrop Measurements
By Mahnaz
| Sample | Concentration (ng/µL) |
|---|---|
| PCR fragment GFP 11 clone 6 | 187.23 |
| PCR fragment GFP 11 clone 8 | 156.85 |
| PCR fragment FRB clone 4 | 75.67 |
| PCR fragment FRB clone 9 | 246.41 |
| PCR fragment GFP 1.9 | 22.06 |
