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
|
Results of PCR products (Gblocks 1.1, 2.1, 3.1, GFP) electrophoresis
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.
gBlock 1.1, 3.1 and GFP1-9 insertion in puc19
By Caroline
The Gblock were inserted once again in puc19 this time after HincII were desactivated in the solution of puc19 digested. Indeed, we noticed that those gBlock contain a hincII restriction sites which explain why we did not obtain PCR amplification with the specific primers.
To carry out this insertion, the usual protocol was made.
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.
