Tuesday 4th October
Lab work
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) were obtained, 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
|
GEL
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, 2 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
|
GEL
The digestion products were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
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
