Thursday 21st July
Lab work
Biobrick characterization
K1372001 from clone 2 digestion
By Alice and Terrence
We made a digestion of K1372001 plasmid following this protocol.
We used EcoR1 and Pst1 fast digest enzymes.
After incubation, 3.3 mL of loading dye is added to digestion products.
We put in wells 10µL of DNA ladder and 20 µL of digestion products.
We set the eletrophoresis machine at 100V for 25min.
Digestion products expected were :
|
|
Band size (Bp)
|
| Plasmid
|
1590
|
| Insert
|
2029
|
Migration of digested product of K1372001
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Making Petri dishes of medium LB (with streptomycin or IPTG and Xgal)
By Laetitia
A Petri dish with LB and streptomycin was made with:
- 20 mL of LB agar
- 10µL of streptomycin (initial concentration 100mg/mL)
We added IPTG and Xgal on 4 Petri dishes of LB and Ampicillin.
For each Petri dish:
- 500µL of water
- 1µL of IPTG
- 1µL of Xgal
The solution was spread on Petri dish
Liquid culture of pcl TAA, TAG and Tq
By Laetitia
2 clones of each petri dish were soaked in a tube of LB and Streptomycin.
6 tubes were made. For each tube:
- 1 mL LB
- 0.5 µL streptomycin
The tubes were incubated at 37°C and 180 rpm overnight.
Visualization
Low Fidelity Dreamtaq PCR of transformed DH5α with pPS16_002, pPS16_004 and pPS16_007
By Mathilde
The transformed pPS16_007 in DH5α put in culture the day before showed blue colonies for the clones 5 and 11. Thus those clones can not be used for high fidelity PCR and be sequenced.
A DreamTaq PCR was made with transformed cultures pPS16_002, pPS16_004 and pPS16_007 following the usual protocol with Tm at 57°c and 5min for the initial denaturation.
We divided up the PCR mix in 18 PCR tubes and added in each one a different clone from transformed each of our three cultures.
The picked colonies were re-plated on a petri dish medium LB + Ampicillin (50µg/mL) + xGal/IPTG (1/1000).
Results :
PCR products expected were :
| Plasmid
|
pPS16_002
|
pPS16_004
|
pPS16_007
|
| Bande Size pb
|
960
|
808
|
706
|
The electropheresis on agarose gel showed good size product PCR for pPS16_004 clones 2 to 6, for pPS16_002 clone 5, and pPS16_007 clones 2 and 3.
Get DNA Closer
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