Monday 26th September
Lab work
Visualization
Colony PCR of 16 clones containing FKBP - GFP 10 in pSB1C3 (pPS16_018)
By Maxence, Mahnaz & Coline
A colony PCR was done for 16 clones (8 clones obtained by Gibson with 2 fragments and 8 clones obtained by Gibson with 3 fragments) from the 23rd September. For that purpose, 16 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)
|
757
|
GEL
NanoDrop Measurements
By Maxence, Manhaz & Caroline
| Sample
|
Concentration (ng/µL)
|
| GFP 1.9 in pSB1C3 (pPS16_020) clone 3
|
8
|
| GFP 1.9 in pSB1C3 (pPS16_020) clone 4
|
14.5
|
| GFP 1.9 in pSB1C3 (pPS16_020) clone 7
|
21.5
|
| GFP 1.9 in pSB1C3 (pPS16_020) clone 8
|
13
|
| PCR product containing FKBP - GFP 10 from the 22nd September
|
38.19
|
| Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 2
|
235
|
| Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 7
|
51
|
| Corrected dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) clone 8
|
200
|
Samples preparation for sequencing
"By Maxence, Mahnaz & Caroline"
20 µL of plasmids dCas9 ST - GFP 11 in pSB1C3 (pPS16_017) (clones 2, 7 and 8) were sent to be sequenced. 20 µL of the primers iPS168 (5µM) and iPS169 (5µM) were sent for sequencing.
By Maxence, Manhaz & Caroline
4 µL of extracted plasmids and 4 µL of DNA ladder were placed in wells and migrated at 100V during 30 min, in odrder to verify the DNA quantity.
GEL
