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EPF-Lausanne/13 August 2009
From 2009.igem.org
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Wet Lab
We re-did a 1.5-step PCR (all LovTap constructs) but this time we prepared 3 reactions of each to purify then in one column so that we'd obtain enough DNA.
OD of the cells incubated with tetracycline was checked after more or less 21h (since inoculation) but the values were all less than 0.1 so probably the tetracycline concentrations were still too high.
Miniprep of the overnight cultures of RFP alone, RFP for the readout system.
PCR of LovTap alone + LovTap-Term with the new primers to correct the mistake at the beginning of the sequence. Then purification of the products, digestion, phosphatase treatment and overnight ligation.
Purified products of 1.5-step PCR and then digested and ligated these as well.
Colony PCR of the clones resulting from yesterday's transformation: the GFP-RBS gave nothing, but for the read-out system 2 4 clones out of 11 seem to have our whole construct =) these were put in liquid culture.
Gel extraction of BBa_E1010 digested backbone which we hadn't treated with phosphatase.
People in the lab
Nath, Basile, Gab, Christian
