IGEM:Groningen/Notebook/iGEM 2011/2011/08/30: Difference between revisions
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== | ==30-8-11== | ||
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<br> Colony PCR of PBAD-RBS-GFP-DT | |||
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<br> Taq 10× buffer: 20μl | |||
<br> dNTPs 10mM: 4μl | |||
<br> MgCl2: 12μl | |||
<br> Forward Biobrick primer: 4μl | |||
<br> Reverse Biobrick primer: 4μl | |||
<br> Taq DNA polymerase: 1μl | |||
<br> MQ water: 155μl | |||
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<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 2,5 min. | |||
<br> Final extension: 72°C for 10 min. | |||
<br> Store infinite at 4°C | |||
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<br> Analyse on a 1% TBE agarose gel. | |||
<br> Transformants seem to contain PBADaraC, but not RBS-GFP-DT since the fragment has a size of 1500 a 1600 bp. | |||
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<br> Overnight cultures PhybB-cI-LVA-DT colonies 2 and 3 and PhybB-LasR-LVA- colonies 2 and 6(with RBSes xD)grew. | |||
<br> Plasmid prep was done. | |||
<br> ND1000- Nanodrop measurements results: DNA concentrations were 30-40 ng/μl | |||
<br> Samples were send for sequencing and glycerol stocks were made | |||
<br> Measurements with FACS were done for PhybB-RBS-GFP, Olesja continues. Also PhybB-RBS-GFP-DT in E.coli BW strain | |||
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<br> Protocol obtained again for gDNA isolation with magnetic beads for report | |||
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<br> New cloning strategy for PBAD-cI-LVA-DT and PBAD-LasR-LVA-DT | |||
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Revision as of 06:49, 30 August 2011
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30-8-11
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