Tuesday 4th October
Visualization
Culture of FRB - GFP 11 in pSB1C3 (pPS16_019) clone 4, FKBP - GFP 10 in pSB1C3 (pPS16_018) clone 6 and GFP 1.9 in pUC19 (pPS16_009) clone 1
By Maxence
As no clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) were obtained and as the concentrations of plasmids extracted the 2nd October were too low, the following clones were put on 2 x 10 mL liquid cultures (LB + 30 µg/mL Cm) grown at 37°C overnight:
- pPS16_018 (FKBP - GFP 10) clone 6
- pPS16_019 (FRB - GFP 11) clone 4
By Maxence
As no good clones were obtained for GFP 1.9 in pSB1C3 (pPS16_020), a new PCR was run using extracted plasmids containing GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October. For each amplification, two matrix were used as two liquid cultures were set the 1st October.
For each 50μl of reaction, mix the following reagents :
- 2 µL of matrix
- 1 µL of dNTPs (10mM)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer (5X)
- 0,5 µL of Phusion polymerase
- 30 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
|
| 60°C
|
30sec
|
| 72°C
|
30 sec
|
| Final Extension
|
72°C
|
2min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
GFP 1.9 in pUC19 (pPS16_009) extracted the 2nd October
|
| Primers
|
iPS84 and iPS140
|
5 µL of PCR products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.
PCR products expected were :
| PCR products
|
Expected band size (bp)
|
| GFP 1.9
|
862
|
Cloning of GFP 1.9 PCR product in pSB1C3 by digestion-ligation
By Maxence
For that purpose, GFP 1.9 PCR product from today was cut by restriction enzymes PstI & XbaI as following:
For the most concentrated one (GFP 1.9 - 1):
- 10 µL of GFP 1.9 PCR product
- 4 µL of buffer FD
- 1 µL of restriction enzyme PstI
- 1 µL of restriction enzyme XbaI
- 24 µL of water
For the less concentrated one (GFP 1.9 - 2):
- 20 µL of GFP 1.9 PCR product
- 4 µL of buffer FD
- 1 µL of restriction enzyme PstI
- 1 µL of restriction enzyme XbaI
- 14 µL of water
Furthermore, BbaB0015 plasmid (in order to have pSB1C3) 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 BbaB0015 plasmid
- 4 µL of buffer FD
- 1 µL of restriction enzyme EcoRI
- 1 µL of restriction enzyme XbaI
- 1 µL of alkaline phosphatase FastAP
- 13 µL of water
The mix were incubated for 1 hour at 37°C. Then, 5 µL of each digestion products and 20 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 (GFP 1.9 PCR product treated by PstI & XbaI)
|
900
|
| Vector digested (BbaB0015 treated by PstI & XbaI)
|
2000
|
The digestion products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol. After cleaned-up, sample from extract 1 and sample from extract 2 were pooled together. Then 2 µL of each cleaned-up products and 20 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
Once template and vector were cut and purified, DNA ligase was used to join the sticky ends of the template and vector together:
- 9 µL of template (GFP 1.9 PCR product treated by PstI & XbaI)
- 3 µL of vector (BbaB0015 treated by PstI & XbaI)
- 2 µL of Buffer T4 10X
- 1 µL of ligase T4 enzyme
- 5 µL of water
A control was done without template. The mix were incubated for 1 hour at rooming temperature. Then the ligase was desactivated at 75°C for 10 minutes.
Transformation of DH5a cells with and GFP 1.9 in pSB1C3 (pPS16_020) obtained by Digestion-Ligation
By Maxence
Dh5a cells were transformed with pSB1C3 containing GFP 1.9 (pPS16_020) or controls (digested plasmid) using the usual protocol.
