Thursday 14th October
Visualization
PCR of GFP 1.9 in pUC19 (pPS16_009) and GFP 1.9 in gblock
By Sylvie
A new PCR was run in order to obtain a new GFP 1.9 PCR product. For each amplification, two matrix were used: GFP 1.9 in pUC19 (pPS16_009) and GFP 1.9 in gblock.
For each 50μl of reaction, mix the following reagents :
- 1 µL of matrix (diluted 10X)
- 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
|
5min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
GFP 1.9 in pUC19 (pPS16_009) or GFP 1.9 in gblock
|
| Primers
|
iPS84 and iPS140
|
By Sylvie
A PCR was run on 24 extracted plasmids pPS16_020 in order to verify if the clonage worked.
For each 50μl of reaction, mix the following reagents :
- 1 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
- 31.5 µL of nuclease free water
Mix gently and aliquot 50 μl of the mix into the PCR tubes on ice.
Perform PCR as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
95°C
|
5min
|
| 30 cycles
|
95°C
|
30sec
|
| 48°C
|
30sec
|
| 72°C
|
30 sec
|
| Final Extension
|
72°C
|
5min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
GFP 1.9 in pSB1C3 (pPS16_020) extracted the 13th October
|
| Primers
|
iPS168 and iPS169
|
Gel of PCR products
By Sylvie
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 (iPS84 & iPS140)
|
862
|
| GFP 1.9 (iPS168 & iPS169)
|
1135
|
No good clones were obtained for GFP 1.9 in pSB1C3 (pPS16_020).
Digestion of FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021), GFP 1.9 PCR product and linearized pSB1C3
Linearized pSB1C3 was digested by restriction enzymes XbaI & PstI:
- 4 µL of plasmid
- 1 µL of buffer FD
- 0.5 µL of restriction enzyme XbaI
- 0.5 µL of restriction enzyme PstI
- 2 µL of water
GFP 1.9 PCR product was digested by restriction enzymes XbaI & PstI:
- 5 µL of GFP 1.9 PCR product
- 1.5 µL of buffer FD
- 1 µL of restriction enzyme XbaI
- 1 µL of restriction enzyme PstI
- 6.5 µL of water
FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) was digested by restriction enzymes SpeI & PstI:
- 10 µL plasmid
- 2 µL of buffer FD
- 1 µL of restriction enzyme SpeI
- 1 µL of restriction enzyme PstI
- 6 µL of water
The mix were incubated for 30 minutes at 37°C. Then, 2 µL of digestion products and 15 µL of DNA ladder were placed in wells and migrated at 100V during 30 min.
Ligation of GFP 1.9 PCR product with PCR blunt
DNA ligase was used to join the sticky ends of the template and vector together. For that purpose, two templates were used: GFP 1.9 PCR product obtained with pUC19 and GFP 1.9 PCR product obtained with gblock.
- 1 µL of vector
- 3 µL of GFP 1.9 PCR product
- 2 µL of Buffer T4 10X
- 1 µL of ligase T4 enzyme
- 3 µL of water
The mix were incubated for 30 minutes at rooming temperature.
Transformation of DH5a cells with GFP 1.9 ligated to PCR blunt
By Sylvie
Dh5a cells were transformed with GFP 1.9 ligated to PCR blunt (two templates for GFP 1.9) or controls (digested plasmid) using the usual protocol.
Co-transformation of DH5a cells with FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021) and GFP 1.9 in pUC19 (pPS16_009)
By Sylvie
Dh5a cells were co-transformed with pSB1C3 containing FRB - GFP 11 - FKBP - GFP 10 (pPS16_021) and with pUC19 containing GFP 1.9 (pPS16_009) using the usual protocol. For that purpose, petri dishes used contained LB + Amp + Cm and two concentration of plasmids were used: 1 µL of plasmids not diluted and 1 µL of plasmids diluted 10X.
Glycerol stocks of clones containing FRB - GFP 11 - FKBP - GFP 10 in pSB1C3 (pPS16_021)
By Sylvie
The glycerol stock of the bacteria with the following plasmids were made.
- pPS16_021 (FRB - GFP 11 - FKBP - GFP 10)
