Team:Paris Saclay/Notebook/September/9
Contents
Friday 9th September
Lab work
Visualization
Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3
Maxence
Colonies were obtained for the Gibson done the 7th September but not for the one done the 8th September. A colony PCR was done for 16 clones from the 7th September. For that purpose, 16 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, 4.13 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 |
| Tm | 30 sec | |
| 72°C | t | |
| Final Extension | 72°C | 7 min |
| Hold | 4°C | $\infinity\$ |
Primers used were:
| Matrix | Clones containing FRB - GFP 11 in pSB1C3 |
|---|---|
| Primers | iPS83 and iPS84 |
| Tm | 61°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 - GFP 11 in pSB1C3 | 730 |
GEL 6
All PCR products were at the good size and seemed to be concentrated. It could be explained as these clones were grown for 2 days. Clones 7, 9, 15 and 16 were selected and were grown at 37°C overnight.
PCR on GFP 1.9 in pUC19, gblock 1.1, gblock 1.2, gblock 2.1 and gblock 2.2 with 3% DMSO
By Maxence
As the annealing temperature (Tm) seems too high to obtain good results for these amplifications, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, PCR was performed on plasmids with the following protocol.
For each 50μl of reaction, mix the following reagents :
- 1 µL of plasmid
- 1 µL of dNTPs (10mM)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer (5X)
- 0,5 µL of Phusion polymerase
- 31 µL of nuclease free water
- 1.5 µL of DMSO
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice. Perform PCR as follow:
| Step | Temperature | Time |
|---|---|---|
| Initial denaturation | 98°C | 30sec |
| 30 cycles | 98°C | 10sec |
| Tm | 30sec | |
| 72°C | t | |
| Final Extension | 72°C | 2min |
| Hold | 4°C | $\infty$ |
Primers used were:
| Matrix | gblock 2.2 in pUC19 |
|---|---|
| Primers | iPS140 and iPS84 |
| Tm | 60°C |
| t | 30 sec |
PCR Clean-up of PCR products
By Maxence
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
NanoDrop Measurements
By Maxence
| Sample | Concentration (ng/µL) |
|---|---|
| PCR fragment FKBP clone 3 | 222.57 |
| PCR fragment FKBP clone 4 | 219.99 |
| PCR fragment FKBP clone 5 | 376.2 |
| PCR fragment gblock 2.2 | 276.33 |
Gel of cleaned up PCR products
By Maxence
After amplification, 3 µL of each cleaned up 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) |
|---|---|
| FKBP | X |
| gblock 2.2 | X |
| GFP 1.9 | 862 |
GEL 4 : FKBP
All PCR products were at the good size.
GEL 5 : gblock 2.2
The PCR product was at the good size.
GEL 6 : gfp 1.9
