IGEM:Groningen/Notebook/iGEM 2011/2011/08/17

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17-8-11 Plasmid prep of taRNA-pSB1A3-DT and RBS-GFP-DT (again) DNA concentrations were around 30ng/μl. De taRNA-pSB1A3-DT were send for sequencing and glycerol stocks were made. Plasmidpreps of RBS-GFP-DT and taRNA-pSB1A3-DT (1 and 4) were used for vector Checked sequencing results PBAD/araC-pSB1C3, were alright!:) Digestion: RBS-GFP-DT 5μl vector 1μl EcoRI 1μl XbaI 3μl Fast digest buffer 1μl FastAP 19μl MQ taRNAc1-pSB1A3-DT 7.5μl vector 1μl EcoRI 1μl XbaI 3μl Fast digest buffer 1μl FastAP 16.5μl MQ taRNAc1-pSB1A3-DT 5μl vector 1μl EcoRI 1μl XbaI 3μl Fast digest buffer 1μl FastAP 19μl MQ PBAD 5μl insert 1μl EcoRI 1μl SpeI 2μl Fast digest buffer 11μl MQ PhybB 4μl insert 1μl EcoRI 1μl SpeI 2μl Fast digest buffer 12μl MQ Incubate for 1 hour at 37 degrees After incubation: DNA clean up with PCR purification kit of Roche Ligation: PBAD/araC-RBS-GFP-DT 8.5 μl vector 5.7 μl insert 1μl T4 DNA ligase 2μl T4 DNA ligase buffer 2.8μl MQ PhybB-taRNA-pSB1A3-DT 8.5 μl vector 6.5 μl insert 1μl T4 DNA ligase 2μl T4 DNA ligase buffer 2μl MQ Self ligation control: 8.5 μl vector 1μl T4 DNA ligase 2μl T4 DNA ligase buffer 8.5μl MQ incubate the samples for 40 minutes at room temperature For transformation: take a sample of the plasmid PhybB-RBS-GFP-DT (5μl of the plasmid) and transform this in TOP10 competent cells in parallel with the other samples. This will be done because of the DH5alpha strain does not grow well in M9 medium for flow cytometry measurements... Transformation: Add to 40μl competent cells 10μl ligation mixture Incubate for 30 min on ice Heat shock: 45s at 42 degrees Incubate cells on ice for 2 min Add 1ml LB medium+ 25mM glucose Incubate cells for 1h/1.5h at 37 degrees Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates Put the plates in the stove at 37 degrees overnight Also, in the morning, the plate with PhybB-RBS-GFP looked alright, had single colonies, so I let them grow this morning in 30 degrees for induction of the PhybB promotor. After 1 and 3 hours, no fluorescent colonies were seen on the plate.