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
|
GEL SYLVIE 7
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.
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:
- 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.
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)
