IGEM:Groningen/Notebook/iGEM 2011/2011/08/01: Difference between revisions
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== | ==1-8-11== | ||
<br> PBAD sample seems to be wrong. Received new one from iGEM Groningen 2009 team. | |||
<br> PCR PBADaraC: | |||
<br> 10× Pfu buffer MgSO4: 5μl | |||
<br> dNTPs 10mM: 1μl | |||
<br> Forward primer 36F: 1μl | |||
<br> Reverse primer 37R: 1μl | |||
<br> (primers contain prefix and suffix) | |||
<br> template: 1μl | |||
<br> pfu polymerase: 1μl | |||
<br> MQ: 40μl | |||
<br> | |||
<br> Checking PCR products on 1% agarosegel | |||
<br> PCR clean up with kit and measure DNA concentration with ND1000 nanodrop. | |||
<br> | |||
<br> Digestion: | |||
<br> | |||
<br> cI-LVA-DT | |||
<br> vector: 2μl | |||
<br> EcoRI: 1μl | |||
<br> XbaI: 1μl | |||
<br> FastAP: 1μl | |||
<br> Fast digest buffer: 3μl | |||
<br> MQ: 22μl | |||
<br> | |||
<br>LasR-LVA-DT | |||
<br> vector: 3μl | |||
<br> EcoRI: 1μl | |||
<br> XbaI: 1μl | |||
<br> FastAP: 1μl | |||
<br> Fast digest buffer: 3μl | |||
<br> MQ: 21μl | |||
<br> | |||
<br> RBS-GFP-DT | |||
<br> vector: 3μl | |||
<br> EcoRI: 1μl | |||
<br> XbaI: 1μl | |||
<br> FastAP: 1μl | |||
<br> Fast digest buffer: 3μl | |||
<br> MQ: 21μl | |||
<br> | |||
<br>PBADaraC | |||
<br> insert: 4μl | |||
<br> EcoRI: 1μl | |||
<br> SpeI: 1μl | |||
<br> Fast digest buffer: 2μl | |||
<br> MQ: 12μl | |||
<br> Digest for 1h at 37 degrees | |||
<br> DNA clean up | |||
<br> | |||
<br> Ligation: | |||
<br> PBADaraC-cI-LVA-DT | |||
<br> Vector: 8.5μl | |||
<br> insert: 6.1μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> MQ: 2.4μl | |||
<br> | |||
<br> PBADaraC-LasR-LVA-DT | |||
<br> Vector: 8.5μl | |||
<br> insert: 6.1μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> MQ: 2.4μl | |||
<br> | |||
<br> PBADaraC-RBS-GFP-DT | |||
<br> Vector: 8.5μl | |||
<br> insert: 6.1μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> MQ: 2.4μl | |||
<br> | |||
<br> Selfligation tests: | |||
<br> Vector: 8.5μl | |||
<br> T4 DNA ligase: 1μl | |||
<br> T4 DNA ligase buffer: 2μl | |||
<br> MQ: 8.5μl | |||
<br> | |||
<br> Ligate for 30 min at room temperature | |||
<br> Transformation as usual | |||
<br> | |||
<br> | |||
Revision as of 07:51, 1 August 2011
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