Thursday 15th September
Lab work
Visualization
Linearization of cleaned-up PCR product pSB1C3
By Maxence
Cleaned-up PCR product pSB1C3 has been linearized for further Gibson application by using DpnI treatment :
- 30 µL of cleaned up PCR product GFP 11 - pSB1C3
- 4 µL of fast digest buffer
- 1 µL of DpnI
- 5 µL of water
The mix was placed 10 minutes at 37°C and was cleaned by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Clean-up of pSB1C3 treated by DpnI
By Maxence
pSB1C3 treated by DpnI was cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
NanoDrop Measurements
By Maxence
| Sample
|
Concentration (ng/µL)
|
| FKBP amplification product from the 8th September
|
152.53
|
| gblock 2.2 amplification product from the 8th September
|
190.02
|
| pSB1C3 treated by DpnI
|
56.75
|
Gibson of cleaned up PCR products FKBP from 8th September x gblock 2.2 from the 8th September x pSB1C3 treated by DpnI
By Maxence
Gibson was performed with cleaned up PCR products FKBP (insert 1) (obtained the 8th September) x gblock 2.2 (insert 2) (obtained the 8th September) x pSB1C3 treated by DpnI (plasmid) with the following protocol:
For each 20μl of reaction, mix the following reagents :
- 0.31 µL of insert 1
- 0.2 µL of insert 2
- 2 µL of plasmid
- 7.49 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 FKBP - GFP 10 in pSB1C3 (pPS16_018) obtained by Gibson
By Maxence & Mahnaz
Dh5a cells were transformed with pSB1C3 containing FKBP - GFP 10 (pPS16_018), or controls (no buffer mix and plasmid alone) using the usual protocol.
Q5 PCR of NM sgRNA in pJET
By Maxence
As we did not achieve to obtain PCR products of NM sgRNA with Phusion, another strategy was tried with Q5. Q5 PCR was performed on plasmids with the following protocol:
For each 50μl of reaction, mix the following reagents :
- 1 µL of plasmid
- 1 µL of dNTPs (10mM)
- 1 µL of each primer mix (10µM)
- 10 µL of Q5 buffer (5X)
- 0,5 µL of Q5 high fidelity polymerase
- 35,5 µL of nuclease free water
Furthermore, 3% DMSO was also used: instead of 35,5 µL of nuclease free water : 1,5 µL of DMSO + 34 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
|
98°C
|
30sec
|
| 5 cycles
|
98°C
|
10sec
|
| 66°C
|
30sec
|
| 72°C
|
20sec
|
| 25 cycles
|
98°C
|
10sec
|
| 72°C
|
30sec
|
| 72°C
|
20sec
|
| Final Extension
|
72°C
|
2min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
gblock NM sgRNA in pJET
|
| Primers
|
iPS157 and iPS158
|
PCR Clean-up of PCR products
By Maxence
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
Gel of cleaned up PCR products
By Maxence
After amplification, 3 µL of cleaned up PCR product 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)
|
| gblock NM sgRNA
|
362
|
