Thursday 25th August
Visualization
pPS16_014 (SgRNA Nm) clone 5, Gibson Assembly's Product (Fragment3-4) clone 6, and pPS16_009 clone 4 extraction
By Terrence
The extraction was carried out following the usual protocol
The Gibson Assembly's Product (Fragment3-4) clone 6 and pPS16_009 clone 4 extraction products were digested with the restriction enzymes BBaI and PSTI
Colony PCR of 12 colonies containing plasmid pJET coding sg-Nm and 12 colonies containing plasmid pJET coding FRB
By Mahnaz
Transformed cells which had been grown overnight were used for colony PCR. For that purpose, 12 clones containing sg-Nm and 12 clones containing FRB were screened and used for the usual protocol of Colony PCR. The same colonies were also plated on Petri dish containing solid LB + 50 µg/mL Amp and liquid cultures (LB + 50 µg/mL Amp) 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)
- 0.5 µL of each primer(10µM)
- 0.13 μl of DreamTaq Pol
PCR was performed as follow:
| Step
|
Temperature
|
Time
|
| Initial denaturation
|
95°C
|
3 min
|
| 25 cycles
|
94°C
|
30 sec
|
| Tm
|
30 sec
|
| 72°C
|
t
|
| Final Extension
|
72°C
|
5min
|
| Hold
|
4°C
|
$\infinity\$
|
Primers used were:
| Matrix
|
plasmid pJET coding sg-Nm
|
plasmid pJET coding FRB
|
| Primers
|
pJET R and pJET F
|
pJET R and pJET F
|
| Tm
|
60.0C
|
60.0C
|
| t
|
15 sec
|
15 sec
|
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)
|
| FRB
|
374
|
| sg-Nm
|
362
|
We can't see anything, the only visibles strands are the priers sequence, The result is not satisfying
Q5 high fidelity PCR of ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004
By mathilde
Fragment 1 (pPS16_001-pPS16_002) and fragment 2 (pPS16_003-pPS16_004E) were each tested three times (A,B,C) according to the following protocol :
- 10µL of Q5 buffer
- 1µL of dNTPs (10mM)
- Primers mix (1µL each)
- 1µL of ligation product
- 0,25 µL of Q5 high fidelity polymerase
- 37,75 µL of nuclease free water
The primers used were IPS83 and IPS 122 for the fragment 1, and IPS 84 and 123 for the fragment 2.
The PCR programm featured 30s of initial denaturation, 72°c of annealing temperature for 30s and 2min of final extension.
Gel electrophoresis of Q5 PCR ligation products pPS16_001-pPS16_002 and pPS16_003-pPS16_004
PCR products were put to migration following the usual protocol.
| PCR products
|
Expected band size (bp)
|
| pPS16_001-pPS16_002
|
1920
|
| pPS16_003-pPS16_004
|
1831
|
Only pPS16_001-pPS16_002 ligation product shows strands at the expected size.
Ligation of pPS16_003-pPS16_004
By Mathilde
- 4 µL of pPS16_003 plasmid
- 4 µL of pPS16_004 plasmid
- 1µL of T4 Buffer 10X
- 1µL of T4 Ligase
The solution is put in incubation at 4°c over night.
