Friday 9th September
Lab work
Visualization
Colony PCR of 16 clones containing FRB - GFP 11 in pSB1C3 (pPS16_019)
By Maxence & Mahnaz
Colonies were obtained for the Gibson done the 7th September but not for the one done the 8th September. A colony PCR was done for 16 clones from the 7th September. For that purpose, 16 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, 4.13 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
|
| 61°C
|
30 sec
|
| 72°C
|
1 min 30 sec
|
| Final Extension
|
72°C
|
7 min
|
| Hold
|
4°C
|
$\infinity\$
|
Primers used were:
| Matrix
|
Clones containing FRB - GFP 11 in pSB1C3 (pPS16_019)
|
| Primers
|
iPS83 and iPS84
|
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 - GFP 11 in pSB1C3
|
730
|
GEL 6
All PCR products were at the good size and seemed to be concentrated. It could be explained as these clones were grown for 2 days. Clones 4, 9, 13 and 16 were selected and were grown at 37°C overnight.
PCR on GFP 1.9 in pUC19, gblock 1.1, gblock 1.2, gblock 2.1 and gblock 2.2 with 3% DMSO
By Maxence & Mahnaz
As the annealing temperature (Tm) seems too high to obtain good results for these amplifications, 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 amplification, two matrix were used: one was a previous PCR products and one was a plasmid.
For each 50μl of reaction, mix the following reagents :
- 1 µL of matrix
- 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
|
30 sec
|
| Final Extension
|
72°C
|
2min
|
| Hold
|
4°C
|
$\infty$
|
Primers used were:
| Matrix
|
PCR product of gblock 1.1 from 2nd September (Q5)
|
gblock 1.1 9/08
|
PCR product of gblock 1.2 from 2nd September (Q5)
|
gblock 1.2 11
|
PCR product of gblock 2.1 from 2nd September (Q5)
|
gblock 2.1 PPS16003
|
PCR product of gblock 2.2 from 2nd September (Q5)
|
gblock 2.2 PPS16004
|
PCR product of GFP 1.9 from 8th September
|
GFP 1.9 in pUC19
|
| Primers
|
iPS140 and iPS120
|
iPS140 and iPS120
|
iPS121 and iPS122
|
iPS121 and iPS122
|
iPS123 and iPS124
|
iPS123 and iPS124
|
iPS125 and iPS84
|
iPS125 and iPS84
|
iPS84 and iPS140
|
iPS84 and iPS140
|
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)
|
| gblock 1.1
|
960
|
| gblock 1.2
|
960
|
| gblock 2.1
|
1023
|
| gblock 2.2
|
808
|
| GFP 1.9
|
862
|
GEL ? : FKBP
All PCR products were at the good size.
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. For each amplification, only PCR products from plasmids were cleaned in order to reduce the risks of mutations. For GFP 1.9 amplification products, the 4 samples were pooled before clean up.
Results about FRB - GFP 11 transformed colonies from 8th September
By Maxence & Mahnaz
During the afternoon, observations were made about the 8th September Gibson, but as results were obtained for the 7th September Gibson, a colony PCR was not run. Nevertheless, different conditions were tested, and a counting of the colonies was done:
| Condition
|
Gibson - usual mix - strain from Philippe
|
Gibson - usual mix - usual strain
|
Gibson - mix from Philippe - strain from Philippe
|
Gibson - mix from Philippe - usual strain
|
Plasmid alone - mix from Philippe - usual strain
|
Plasmid alone - mix from Philippe - strain from Philippe
|
Otherwise
|
| Number of clones
|
2
|
25
|
0
|
$\infty$
|
3
|
2
|
0
|
