EPF-Lausanne/13 July 2009

Wet Lab

Inverter TetR (BBa_I6007) was transformed again. And the new plasmid (created on July the 10th, 10.07.09) LacI-RBS was transformed on DH5-alpha competent cells.

We failed in purifing our Terminator (BBa_B0010), do it again tomorrow, beginning with the digestion, etc.

Cloning Strategy

Restriction enzymes on Biolabs website and clevage oligonucleotides

TRP promoter biobrick strategy

• Problem to overcome:

SpeI sites on Trp promoter sequence and it's an upstream part which has to be cut with ES.

• Strategy:

PCR: Forward primer having E and X sites and Reverse primer NheI.

Digest Trp promoter with E and NheI.

Digest plasmid with E and X.

Ligation -> E site is recreated; X and NheI have compatible ends so ligation is possible and the site is destroyed (mixted site).

People in the lab

Heidi, Tu, Nath, Caro