IGEM:Groningen/2009/Notebook/iGEM 2011/2011/07/05

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Entry title
PCR of HybB promotor, PcI, PlasI, LasR-LVA, cI LVA. PCR protocol: Denaturation: 94 °C 7 minutes Cycle 30× Denaturation: 94 °C 1 minute Annealing: 30 seconds (Temperature gradient: 65 °C hybB, 60 °C PcI and PlasI, 57°C LasR-LVA and cI-LVA) Extension: 2 minutes Final extension: 7 minutes Store infinite at 4 °C

PCR cleanup and vector cleanup according to the High Pure PCR purification kit of Roche (LOT: 11 732 676 001 version 15.0)

Check PCR cleanups at 1.5 % agarose gel: products visible, one band, right size! (During agarose gel electrophoresis nanodrop of samples: Vectors: 50 ng/μl, HybB: 70ng/μl, PcI: 65.9 ng/μl, PlasI: 42.7 ng/μl, cI-LVA: 149.3 ng/μl, LasR-LVA 127.1 ng/μl)

Digestion of PCR products and vectors: One reaction for HybB in pSB1C3: 1μl pSB1C3 0.5μl hybB 1μl EcoRI 1μl PstI 2μl Fast digest buffer 14.5 MQ water HybB: 3μl hybB 1μl EcoRI 1μl SpeI 2μl fast digest buffer 13μl MQ water PcI: 2μl PcI 1μl EcoRI 1μl SpeI 2μl fast digest buffer 14μl MQ water PlasI: 3μl PlasI 1μl EcoRI 1μl SpeI 2μl fast digest buffer 13μl MQ water cI-LVA: 2μl cI-LVA 1μl EcoRI 1μl SpeI 2μl fast digest buffer 14μl MQ water LasR-LVA: 2μl LasR-LVA 1μl EcoRI 1μl SpeI 2μl fast digest buffer 14μl MQ water RBS-GFP-DT vector: 3μl RBS-GFP-DT vector 1μl EcoRI 1μl XbaI 2μl fast digest buffer 13μl MQ water AmpR-DT vector: 3μl RBS-GFP-DT vector 1μl EcoRI 1μl XbaI 2μl fast digest buffer 13μl MQ water Digest for 30 minutes at 37°C in a waterbath. After digestion: PCR cleanup and vector cleanup according to the High Pure PCR purification kit of Roche (LOT: 11 732 676 001 version 15.0), only dilute the hybB-pSB1C3 in 17μl to use directly for ligation. Calculate the amount of insert which is required for 8.5μl of vector (=20ng) with the ligation calculator (http://www.insilico.uni-duesseldorf.de/Lig_Input.html) and make your own calculation for the dilution of your insert Ligation: HybB in RBS-GFP-DT vector: 5μl hybB 8.5μl RBS-GFP-DT vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 3.5μl MQ water PcI in RBS-GFP-DT vector: 2μl PcI 8.5μl RBS-GFP-DT vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 6.5μl MQ water PlasI in RBS-GFP-DT vector: 3μl PlasI 8.5μl RBS-GFP-DT vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 2.5μl MQ water cI-LVA in ampR DT vector: 6μl cI-LVA 8.5μl ampR-DT vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 2.5μl MQ water LasR-LVA 7μl LasR-LVA 8.5μl RBS-GFP-DT vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 1.5μl MQ water Self ligation testing of the vectors: 8.5μl vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 9.5 μl MQ water Incubate at room temperature for 30 minutes Competent cells were prepared with the protocol of openwetware: Transformation of E.coli DH5 alpha competent cells protocol: http://openwetware.org/wiki/Transforming_chemically_competent_cells * N.B: 10μl ligation mixture was used for each transformation


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