IGEM:Groningen/Notebook/iGEM 2011/2011/09/02

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2-9-11 Plasmid Prep of overnight cultures: cI-LVA and LasR-LVA in chloramphenicol vector+ K077002 and K07004 for cI production. According to the ND1000- Nanodrop, DNA concentration was: around 30ng/μl Use the cI-LVA and LasR-LVA vectors for digestion Digestion: PBAD 5.6 μl PBAD PCR product 1μl EcoRI 1μl SpeI 2μl FD buffer 10.4μl MQ water cI-LVA vector: 4μl cI-LVA vector 1μl EcoRI 1μl XbaI 3μl FD buffer 1μl FastAP 20μl MQ water LasR-LVA vector: 4μl LasR-LVA vector 1μl EcoRI 1μl XbaI 3μl FD buffer 1μl FastAP 20μl MQ water pSB1C3-DT vector: 3μl pSB1C3-DT 1μl EcoRI 1μl XbaI 3μl FD buffer 1μl FastAP 21μl MQ water pSB1C3-DT vector 3μl pSB1C3-DT 1μl XbaI 1μl PstI 3μl FD buffer 1μl FastAP 21μl MQ water LasR-LVA PCR product: 4μl LasR-LVA PCR product 1μl XbaI 1μl PstI 2μl FD buffer 12μl MQ water Incubate the samples for 1h at 37°C After digestion: DNA clean up with the High Pure PCR Purification kit of Roche Ligation: PBAD-cI-LVA 5μl PBAD 8.5μl cI-LVA vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 3.5μl MQ water PBAD-LasR-LVA 5μl PBAD 8.5μl LasR-LVA vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 3.5μl MQ water PBAD-pSB1C3DT 7μl PBAD 8.5μl pSB1C3DT vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase 1.5μl MQ water LasR-LVA in pSB1C3DT vector 8.5μl LasR-LVA PCR product 8.5μl pSB1C3DT vector 2μl T4 DNA ligase buffer 1μl T4 DNA ligase Ligate for 30-40 minutes at room temperature Transformation: Add to 40μl competent E.coli DH5alha cells 10μl ligation mixture Incubate the samples for 30 min. on ice Heat shock: Incubate the samples for 45s at 42°C After the heat shock: place the cells for 2 min on ice and add after the incubation time on ice 1ml of LB+25mM glucose. Incubate the samples for 1h to 1.5 h at 37°C Plate the cells out: Centrifuge the sample, leave 100μl of LB+glucose in the tube and resuspend the pellet. Plate 90μl and 10μl of the resuspended pellet on plates containing antibiotic(s) Colony PCR Colony PCR of the RBS-GFP-DT plasmid from the distribution kit. Yesterday, that sample had been transformed again, since there were some difficulties with the plasmid in cloning purposes. It did not seem to have the insert (RBS-GFP-DT) anymore. PCR mix: 10× Taq buffer: 20μl dNTPs 10mM each: 4μl MgCl2: 12μl BB forward primer: 4μl BB reverse primer: 4μl Taq polymerase: 1μl MQ water: 155μl PCR conditions: Pre heated lid at 111°C Denaturation: 94°C for 10 min Cycle 33× Denaturation: 94°C for 30s Annealing: 60°C for 30s Extension: 72°C for 2.5 min Final extension: 72°C for 10 min Store at 4°C infinite Analyse on a 1% TBE agarose gel