Tuesday 26th July
Lab work
Visualization
By Alice
PCR peformed on July 25 with bacteria containing plasmids pPS16_001, pPS16_005 and pPS16_009 did not give us expected results. We performed again this PCR, adding bacteria transformed with pPS16_003 (clone 3) that got good sequencing results. The PCR with Q5® High-Fidelity 2X Master Mix was performed on these clones following this protocol. Specific primers were used for each plasmids (table below). Annealing temperature calculated was 72°C. After amplification, 1 µL of loading dye was added to 5µL of each PCR products. Then 5 µL of this mix were put into a well of the gel. A last well contained 10µL of DNA ladder. PCR products were migrated 25min at 100V.
PCR products expected were :
| Plasmids
|
gBlocks
|
Primer Forward
|
Primer Reverse
|
Band size (bp)
|
| pPS16_001
|
1.1
|
iPS138
|
iPS120
|
960
|
| pPS16_003
|
2.1
|
iPS123
|
iPS124
|
1023
|
| pPS16_005
|
3.1
|
iPS138
|
iPS126
|
960
|
| pPS16_009
|
GFP 1-9
|
iPS138
|
iPS139
|
862
|
Low Fidelity Dreamtaq PCR of DH5α|pPS16_002
By Laetitia
We made a DreamTaq PCR on DH5α|pPS16_002 transformed cultures containing the Gblock 1.2.
The PCR was done on 6 clones of transformed cells: 4 clones of the Petri dish from 19/07 and 2 clones of the Petri dish from 22/07.
The usual protocol was used with TM at 57°C.
These 6 clones were re-plated on a Petri dish containing LB, Ampicilin, IPTG and X-Gal.
Biobrick Characterization
BL21 electrocompetent cells preparation and transformation
By Léa, Charlène and Sylvie
We did an electro-transformation of BL21 with two solutions of glycerol different because we suspected that our solution where the root of our problem of transformation.
iGEM team's glycerol :
- pcl_TAA + K1372001 (time constant equal to 5.8ms)
- pcl_TAG + K1372001 (time constant equal to 6,2 ms)
- pcl_Tq + K1372001 (time constant equal to 6 ms)
- K1372001 (time constant equal to 6 ms)
Sylvie team's glycerol:
- pcl_TAA + K1372001 (time constant equal to 6 ms)
- pcl_TAG + K1372001 (time constant equal to 6 ms)
For the third conditions with iGEM team's glycerol, cells were displayed on LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) medium. For each condition, we made a dish with 50µL of cells and another with 500µL of cells. 150µL of BL21|K1372001 were displayed on LB + Chloramphenicol medium.
For BL21 transformed with Sylvie team's glycerol, 150µL were displayed on LB + Streptomycin + Chloramphenicol medium.
Two controls were made with 100µL of BL21 which were displayed on LB + Chloramphénicol or LB + Streptomycin medium.
Cells grew overnight at 37°C.
Culture of BL21 electrocompetent cells
By Léa
A BL21 colony was put into 4mL of liquid LB medium, and grown at 37°C, 180 rpm overnight.
