Tuesday 20th September
Lab work
Visualization
Samples preparation for sequencing
"By Maxence, Mahnaz & Coline"
20 µL of plasmids pSB1C3 FKBP - GFP 10 (clones 7, 8, 9 and 10) were sent to be sequenced. 20 µL of the primers iPS84 (5µM) and iPS140 (5µM) were sent for sequencing.
NanoDrop Measurements
"By Maxence, Mahnaz & Coline"
| Sample
|
Concentration (ng/µL)
|
| FKBP - GFP 10 clone 7
|
X
|
| FKBP - GFP 10 clone 8
|
X
|
| FKBP - GFP 10 clone 9
|
X
|
| FKBP - GFP 10 clone 10
|
X
|
| PCR product 1 obtained by iPS174 & iPS175
|
108.17
|
| PCR product 2 obtained by iPS173 & iPS176
|
136.91
|
Colony PCR of the 4 clones containing GFP 1.9 in pSB1C3 (pPS16_020) from the 12th September
By Maxence, Mahnaz, Coline & Caroline
As results from sequencing were not good, clones sent previously and stocked in glycerol (2, 7, 8 and 12) were put on plate the 19th September, as each colony may not not be homogenous enough. Another colony PCR was done and anothers primers were used.
For that purpose, the 4 clones were 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 GFP 1.9 in pSB1C3 (pPS16_020)
|
| 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)
|
| GFP 1.9 in pSB1C3
|
862
|
GEL
Gibson of cleaned up PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 treated by DpnI
By Maxence, Mahnaz & Coline
Gibson was performed with cleaned up PCR products from pSB1C3 with dCas9 ST - GFP 11 (pPS16_017) clone 8 treated by DpnI, more precisely PCR product 1 obtained by iPS174 & iPS175 (plasmid) and PCR product 2 obtained by iPS173 & iPS176 (insert) with the following protocol:
For each 20μl of reaction, mix the following reagents :
- 0.63 µL of insert
- 1.2 µL of plasmid
- 8.17 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 correct dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) obtained by Gibson
By Maxence, Mahnaz & Coline
Dh5a cells were transformed with corrected pSB1C3 containing dCas9 ST - GFP 11 (pPS16_017), or controls (no buffer mix and plasmid alone) using the usual protocol.
Cloning of GFP 1.9 from pUC19 in pSB1C3 by digestion-ligation
By Maxence, Mahnaz & Coline
4µL of 2.1 purify PCR products, 4µL of 2.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed.
4µL of 3.1 purify PCR products, 4µL of 3.2 purify PCR products, 1µL of ligase T4 Buffer and 1µL of ligase T4 were mixed.
They were incubated for 1h at RT.
