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EPF-Lausanne/8 August 2008
From 2008.igem.org
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Cloning with the ligation products
The ligations done with the part which were not run through a gel yielded some colonies. We took 5 colonies from each of the two ligations which seemed to work (the one with diluted and the one with undiluted vector, as vector concentration is much higher than insert concentration. We plated them anew on Kan-Amp plates and also culture them overnight in LB with the correct antibiotic. The pUC19 control was given same treatment but with Amp resistance only.
Digestions
The digestion which had not worked was redone. We did the B0034 with E/X and the R0040 with E/S. Concentrations were quite low, quality seemed OK. The gel did not let us take the small fragment, so we just took the large fragment and purified it with the Qiagen gel purification kit.
PCR with the paper extracts
In order to obtain the P0140 we try PCRing up from the paper extracts. We try the Lac promoter as well (R0010), I0466 because we know it works so we use it as a positive control, and used the positive control for PCR from the Lab (PCR proven to work), we also use positive controls of other PCRs which had worked before, from the parts which we miniprepped.
2 Step PCR
We purified the PCR product from our 2step PCR from last week. It will be sent to be sequenced next week. We performed the first step of the 2step PCR for LasR from the Prey plasmid, as well as the I1466 (RhlR) and a control. The second step will be done Monday.
