IGEM:Groningen/Notebook/iGEM 2011/2011/07/18: Difference between revisions
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== | ==18-7-11== | ||
<br> Today, Digestion, DNA clean up with High Pure PCR purification kit of Roche, ligation, transformation to create transformants <br> with: PBAD-LasR-LVA-DT and PhybB-LasR-LVA-DT | |||
<br> Also, colony of PBAD-RBS-GFP and PBAD-cI-LVA-DT were grown overnight in LB medium with ampicillin and this morning, the culture had been grown, so plasmid prep, glycerol stock and sample for sequencing should be made. | |||
<br> Digestion: | |||
<br> LasR-LVA-DT vector | |||
<br> 2μl vector | |||
<br> 1μl EcoR1 | |||
<br> 1μl XbaI | |||
<br> 3μl Fast digest buffer | |||
<br> 1μl Fast AP | |||
<br> 22μl MQ | |||
<br> | |||
<br> pBAD | |||
<br> 10μl pBAD | |||
<br> 1μl EcoR1 | |||
<br> 1μl SpeI | |||
<br> 2μl Fast digest buffer | |||
<br> 6μl MQ | |||
<br> | |||
<br> PhybB | |||
<br> 3μl PhybB | |||
<br> 1μl EcoR1 | |||
<br> 1μl SpeI | |||
<br> 2μl Fast digest buffer | |||
<br> 13μl MQ | |||
<br> Incubate the samples at 37°C for 30 min. | |||
<br> Do a DNA purification with the PCR purification kit from Roche | |||
<br> Ligation: | |||
<br> | |||
<br> PBAD-LasR-LVA-DT vector | |||
<br> 8.5μl vector | |||
<br> 6.1μl insert | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 2.4μl MQ | |||
<br> | |||
<br> PhybB-LasR-LVA-DT vector | |||
<br> 8.5μl vector | |||
<br> 4.1μl insert | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 4.4μl MQ | |||
<br> | |||
<br>Self ligation: | |||
<br> 8.5μl vector | |||
<br> 1μl T4 DNA ligase | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 8.5μl MQ | |||
<br> | |||
<br> Ligate at room temperature for 30 min. | |||
<br> | |||
<br>Transform as usual (see 12th of July) | |||
<br> | |||
<br> Plasmid prep of overnight culture construct: LasR-LVA-DT (plasmid backbone with ampicillin resistance) | |||
<br> Elute in 100μl MQ | |||
<br> Use 10μl for gel electrophoresis to check wether a plasmid is present in the sample. Plasmid was seen on a 1% agarose gel with <br>TBE (with the help of gelred) | |||
<br> Nanodrop result: 67.2 ng/μl. | |||
<br> Send for sequencing: 5μl Sample + 5μl primer of 5μM. | |||
<br> Glycerol stock was made: 250μl Glycerol 85% + 750μl bacterial culture (overnight culture) | |||
<br> | |||
<br> Colony PCR with samples PBAD-cI-LVA-DT, PhybB-cI-LVA-DT and PBAD-RBS-GFP-DT (samples ligation mixtures stored overnight in fridge)was done as followed: | |||
<br> Mastermix scheme: | |||
<br> Taq 10× buffer with NH4SO4 without MgCl2: 58.00μl | |||
<br> dNTPs 10mM each: 11.6μl | |||
<br> MgCl2: 34.8μl | |||
<br> Taq polymerase 5u/μl: 2.90μl | |||
<br> Biobrick vector forward primer 10μM: 11.6μl | |||
<br> Biobrick vector reverse primer 10μM: 11.6μl | |||
<br> MilliQ water: 449.50μl | |||
<br> | |||
<br> PCR conditions: | |||
<br> Preheated lid: 111°C | |||
<br> Denaturation: 94°C for 10 min. | |||
<br> Cycle (33×) | |||
<br> Denaturation: 94°C for 30s. | |||
<br> Annealing: 60°C for 30s. | |||
<br> Extension: 72°C for 2 min. | |||
<br> Final extension: 72°C for 10 min. | |||
<br> Store at 4°C infinite | |||
<br> | |||
<br> PCR products were analysed on a 1% agarose gel with TBE. | |||
<br> Results: PBAD-cI-LVA-DT colony 1 seems to be right (correct size on gel (2kb) ) | |||
<br> PhybB-cI-LVA-DT can be right, is difficult to see, maybe use one colony and tomorrow (with other colony PCR probably, pick <br> some of the old plates of this sample aswell. | |||
<br> PBAD-RBS-GFP-DT colony 4 looks alright, but is difficult to see because the gel is not very good, so just try along with the <br> pBAD-cI-LVA no right transformants, but the one of friday will be used as the sample send for sequencing. | |||
<br> | |||
<br>PCR for PhybB PCR product (there is not much left anymore...) | |||
<br> | |||
<br> PCR was done as followed: | |||
<br> Mastermix scheme: | |||
<br> Taq 10× buffer with NH4SO4 without MgCl2: 25.00μl | |||
<br> dNTPs 10mM each: 5μl | |||
<br> MgCl2: 15μl | |||
<br> Taq polymerase 5u/μl: 1.25μl | |||
<br> Pfu polymerase: 0.2μl | |||
<br> Forward primer PhybB: 5μl | |||
<br> Reverse primer PhybB: 5μl | |||
<br> MilliQ water: 193.55μl | |||
<br> End volume in PCR tube: 50μl. | |||
<br> | |||
<br> PCR conditions: | |||
<br> Preheated lid: 111°C | |||
<br> Denaturation: 94°C for 10 min. | |||
<br> Cycle (37×) | |||
<br> Denaturation: 94°C for 30s. | |||
<br> Annealing: 65°C and 70°C (gradient) for 30s. | |||
<br> Extension: 72°C for 2 min. | |||
<br> Final extension: 72°C for 10 min. | |||
<br> Store at 4°C infinite (overnight) | |||
<br> | |||
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Revision as of 07:49, 18 July 2011
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18-7-11
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