Tuesday 26th July
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 on gel. 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 spread on a Petri dish containing LB, Ampicilin, IPTG and X-Gal.
Results of PCR products (Gblocks 1.2) electrophoresis
gBlock 1.1, 3.1 and GFP1-9 insertion in puc19
By Caroline
The gblocks were inserted once again in puc19 this time after HincII were desactivated after puc19 digestion. Indeed, we noticed that those gBlocks 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 electrotransformation of BL21 with two different solutions of glycerol because we suspected that our solution where the root of our problem of transformation.
As we have problem with this electrotransformation, the transformation was made twice this time: once by us and another by the I2BC staff.
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 spread on LB + Streptomycin (50µg/mL) + Chloramphenicol (30µg/mL) medium. For each condition, we made a Petri dish with 50µL of cells and another with 500µL of cells. 150µL of BL21|K1372001 were spread on LB + Chloramphenicol medium.
For BL21 transformed with Sylvie team's glycerol, 150µL were spread on LB + Streptomycin + Chloramphenicol medium.
Two controls were made with 100µL of BL21 which were spread 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.
