Thursday 8th September
Lab work
Visualization
Gibson of cleaned up PCR products FRB from clone 9 x pSB1C3 - GFP 11 from clone 8
By Maxence & Mahnaz
As no colonies were obtained after one night culture, a new 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.
Concentration of insert and plasmid were determinated again by Nano drop. For each 20μl of reaction, mix the following reagents :
- 0.65 µL of insert
- 1.1 µL of plasmid
- 8,25 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). Two different buffer mix were tested (the usual one and another one from Philippe). 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 containing controls (no buffer mix and plasmid alone) using the usual protocol. As mentioned before, two buffer mix were used. Furthermore, two different DH5a strains were tested (the usual one and another one from Philippe) and at least 12 transformants were cultured overnight : 3 (1 Gibson and 2 controls) x 2 buffer mix x 2 DH5a strains.
Q5 PCR on FKBP in pJET and gblock 2.2 (part of dCas9 NM - GFP 10) for new clonage strategy
By Maxence & Mahnaz
An other clonage strategy was performed by using Gibson between FKBP, gblock 2.2 (part of dCas9 NM - GFP 10) and pSB1C3 (the one from dCas9 ST - GFP 11) in order to have FKBP - GFP 10 in pSB1C3 (pPS16_018). For that purpose, 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)
- 2.5 µL of each primer mix (10µM)
- 10 µL of Q5 buffer (5X)
- 0,5 µL of Q5 high fidelity 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
|
98°C
|
30sec
|
| 30 cycles
|
98°C
|
10sec
|
| Tm
|
20sec
|
| 72°C
|
t
|
| Final Extension
|
72°C
|
2min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
gblock FKBP in pJET clones 3, 4 and 5
|
gblock 2.2 in pUC19
|
| Primers
|
iPS145 and iPS146
|
iPS147 and iPS84
|
| Tm
|
62°C
|
70°C
|
| t
|
15 sec
|
15 sec
|
PCR on GFP 1.9 in pUC19 with 3% DMSO
By Maxence & Mahnaz
As the annealing temperature (Tm) seems too high to obtain good results for GFP 1.9 in pUC19 amplification, DMSO was used in order to reduce the annealing temperature during the PCR. For that purpose, 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)
- 2.5 µL of each primer mix (10µM)
- 10 µL of buffer (5X)
- 0,5 µL of Phusion polymerase
- 31 µL of nuclease free water
- 1.5 µL of DMSO
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
|
| 30 cycles
|
98°C
|
10sec
|
| 60°C
|
30sec
|
| 72°C
|
30sec
|
| Final Extension
|
72°C
|
2min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
gblock 2.2 in pUC19
|
| Primers
|
iPS140 and iPS84
|
PCR Clean-up of PCR products
By Maxence & Mahnaz
PCR products obtained were cleaned up by using the NucleoSpin Gel and PCR Clean-up kit protocol.
NanoDrop Measurements
By Maxence & Mahnaz
| Sample
|
Concentration (ng/µL)
|
| PCR fragment FKBP clone 3
|
222.57
|
| PCR fragment FKBP clone 4
|
219.99
|
| PCR fragment FKBP clone 5
|
376.2
|
| PCR fragment gblock 2.2
|
276.33
|
Gel of cleaned up PCR products
By Maxence & Mahnaz
After amplification, 3 µL of each cleaned up 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
|
419
|
| gblock 2.2
|
808
|
| GFP 1.9
|
862
|
Migration of FKBP plasmid
GEL 4 : FKBP
All PCR products were at the good size.
GEL 5 : gblock 2.2
All PCR product were at the good size.
GEL 6 : gfp 1.9
