Thursday 21^st 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 :

Migration of digested product of K1372001

Making Petri dishes of medium LB (with streptomycin or IPTG and X-Gal)

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 X-Gal on 4 Petri dishes of LB and Ampicillin. For each Petri dish:

• 500µL of water
• 1µL of IPTG
• 1µL of X-Gal

The solution was spread on Petri dish

Liquid culture of DH5a transformed with pcl_TAA, pcl_TAG and pcl_Tq

By Laetitia

2 clones of each Petri dish were inoculated 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 DH5α transformed with pPS16_002, pPS16_004 and pPS16_007

By Mathilde

DH5α|pPS16_007 that were 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) + X-Gal/IPTG (1/1000).

Results : PCR products expected were :

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

By