IGEM:Groningen/Notebook/iGEM 2011/2011/07/08

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*8th of July
Colony PCR of transformants to check if any transformant contains the plasmid with cI-LVA and LasR-LVA. 20μl total volume was used and the PCR was done with taq polymerase. PCR program: Denaturation: 95 °C for ten minutes Cycle (33×): Denaturation: 95°C for 30 seconds Annealing:    60°C for 30 seconds Extension:    72°C for 2 minutes Final extension: 72°C for ten minutes Store at 4 °C infinite PCR with the new pBAD primers (dissolved and dilution made by me) PCR was done in total colume of 50μl and with tag and Pfu polymerase (ratio 6:1) so Pfu can correct the mistakes that taq made Denaturation: 94°C for ten minutes Cycle (30×) Denaturation: 94°C for 30 seconds Annealing: 62°C and 65°C for 30 seconds Extension: 72°C for 2 minutes Final extension: 72°C for ten minutes Store at 4 °C infinite Check them on agarose gel 1% (TBE). No right transformants for cI-LVA and LasR-LVA. For next time calculations: Digestion. cI-LVA: use 3μl for digestion with EcoRI and SpeI and LasR-LVA: use 2μl for digestion Ligationm. cI-LVA: use 7.8 of purified sample for ligation and LasR-LVA: use 6.5μl of purified sample for ligation. Digestion of the vector should include FastAP, since so far, there have been only self ligation products. PCR of pBAD: A lot of unspecific binding products. Another PCR was done with an annealing temperature of 67 and 70 degrees.


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