IGEM:Groningen/Notebook/iGEM 2011/2011/08/22: Difference between revisions
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<br> After check, digestion: | <br> After check, digestion: | ||
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<br> ------------ | |||
<br> PCR did not go well. Do an overnight PCR again and everything from digestion to below will be done tomorrow | |||
<br> | |||
<br> PCR on templates with cI-LVA and LasR-LVA: | |||
<br> Composition per reaction: | |||
<br> 10× pfu buffer with MgSO4: 5μl | |||
<br> dNTPs 10mM: 1μl | |||
<br> Forward primer: 1μl | |||
<br> Reverse : 1μl | |||
<br> Template: 1μl | |||
<br> Pfu polymerase: 1μl | |||
<br> MQ water: 40μl | |||
<br> | |||
<br> PCR conditions: | |||
<br> Pre heated lid: 111°C | |||
<br> Hotstart | |||
<br> Denaturation: 94°C for 5min | |||
<br> Cycle (33×) | |||
<br> Denaturation: 94°C for 30s | |||
<br> Annealing: 69°C for 30s | |||
<br> Extenstion: 72°C for 2min | |||
<br> Final extension: 72°C for 10min | |||
<br> Store infinite at 4°C | |||
<br> Analyse PCR samples on a 1% agarosegel | |||
<br> | |||
<br> | |||
<br> | |||
<br> | |||
<br> Digestion | |||
<br> cI-LVA | <br> cI-LVA | ||
<br> 7μl insert | <br> 7μl insert |
Revision as of 10:15, 22 August 2011
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22-8-11
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