Wednesday 7th September
Lab work
Visualization
Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8
By Maxence & Mahnaz
Gibson was performed with cleaned up PCR products FRB from clone 9 (insert) x pSB1C3 - GFP 11 from clone 8 (plasmid) (obtained the 6th September) with the following protocol:
For each 20μl of reaction, mix the following reagents :
- 0.64 µL of insert
- 3.68 µL of plasmid
- 5.67 µL of water
- 10 µL of buffer mix
Two controls were done: one without insert and one without buffer (water was added in order to have a total of 20 µL). The PCR was performed as follow : 1 hour at 50°C.
Transformation of DH5a cells with FRB - GFP 11 in pSB1C3 (pPS16_019) obtained by Gibson
By Maxence & Mahnaz
Dh5a cells were transformed with pSB1C3 containing FRB - GFP 11 (pPS16_019), or controls (no buffer mix and plasmid alone) using the usual protocol.
Colony PCR of 32 clones containing dCas9 NM - GFP 10 in pSB1C3 (pPS16_016)
By Maxence & Mahnaz
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 16 clones containing dCas9 NM - GFP 10 obtained by Q5 amplification and 16 clones containing dCas9 NM - GFP 10 obtained by Phusion amplification 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, 4.13 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
|
| 63.6°C
|
30 sec
|
| 72°C
|
1 min 30 sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infinity\$
|
Primers used were:
| Matrix
|
Clones containing dCas9 NM - GFP 10 in pSB1C3 (pPS16_016)
|
| Primers
|
iPS83 and iPS84
|
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)
|
| dCas9 NM - GFP 10 - pSB1C3
|
3688
|
[File:T--Paris Saclay--090716 Gel3.jpg|400px|thumb|center|Result of the migration]]
GEL 2 + GEL 3
Only PCR products around 650 pb were obtained for both Pol. This kind of results has already been obtained by the past and the experience has to be improved.
Samples preparation for sequencing
By Maxence & Mahnaz
20 µL of plasmides dCas9 ST - GFP 11 (pPS16_016) (clones 6 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM), iPS169 (5µM) and iPS171 (5µM) were sent for sequencing.
