Tuesday 27th September
Visualization
By Maxence & Mahnaz
In order to verify if extracted plasmids contain GFP 1.9, a PCR was run with iPS168 & iPS169 to amplify the insert. For that purpose, plasmids sent for sequencing the 14th September (clones 2, 7, 8 and 12) and plasmids extracted the 23rd Sepembter (clones 3, 4, 7 and 8) were used.
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
- 32.5 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
|
30sec
|
| 30 cycles
|
95°C
|
30sec
|
| 64.4°C
|
30sec
|
| 72°C
|
30sec
|
| Final Extension
|
72°C
|
5min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Extracted plasmid pSB1C3 containing GFP 1.9 (pPS16_020)
|
| Primers
|
iPS168 and iPS169
|
Gel of PCR products
By Maxence & Manhaz
4 µL of PCR products and 4 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
|
1135
|
Plasmids extraction of clones 3, 4, 7 and 8 containing GFP 1.9 in pSB1C3 (pPS16_020)
"By Maxence & Mahnaz"
As th quantity of DNA obtained previously was not enough for sequencing, plasmids were extracted using the "Charge Switch-Pro Plasmid Miniprep" kit:
- pPS16_020 (GFP 1.9) clone 3
- pPS16_020 (GFP 1.9) clone 4
- pPS16_020 (GFP 1.9) clone 7
- pPS16_020 (GFP 1.9) clone 8
Colony PCR of 23 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)
By Maxence & Mahnaz
As the two clones selected yesterday were not grown, a new colony PCR was done for 23 clones (16 clones obtained by Gibson with 2 fragments and 7 clones obtained by Gibson with 3 fragments) from the 23rd September. For that purpose, 23 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
|
30sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
Clones containing FKBP - GFP 10 in pSB1C3 (pPS16_19)
|
| 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)
|
| FKBP - GFP 10 in pSB1C3 (pPS16_019)
|
1030
|
