IGEM:Groningen/Notebook/iGEM 2011/2011/07/20: Difference between revisions
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<br> PCR products were analysed on a 1% agarose gel with TBE. | <br> PCR products were analysed on a 1% agarose gel with TBE. | ||
<br> PCR clean up+ measure DNA concentration with nanodrop: | <br> PCR clean up+ measure DNA concentration with nanodrop. | ||
<br> PCR with BB primers: 125.3 ng/μl, PCR LasR-LVA primers: 89.1μl | |||
<br> Ligation calculator: need 57ng of PCR product (BB) and 46ng of PCR product (LasR-LVA primers) | |||
<br> After calculation: | |||
<br> Need 3.5μl PCR product (BB) for digestion and 6.5μl of the clean up for ligation. Need 4μl of PCR product (LasR-LVA primers) | |||
<br> and 6.5μl for ligation | |||
<br> Digestion: | <br> Digestion: | ||
<br> LasR-LVA | <br> LasR-LVA | ||
<br> . | <br> 3.5 and 4μl insert | ||
<br> 1μl SpeI | <br> 1μl SpeI | ||
<br> 2μl Tango buffer | <br> 2μl Tango buffer | ||
<br> . | <br> 13.5 or 13μl MQ | ||
<br> Total volume (V): 20μl | <br> Total volume (V): 20μl | ||
<br> After 1.5 hours incubation: | <br> After 1.5 hours incubation: | ||
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<br> Ligation: (overnight) | <br> Ligation: (overnight) | ||
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<br>LasR-LVA in pSB1A3-DT | |||
<br>6.5μl insert | |||
<br>8.5μl vector | |||
<br>2μl T4 DNA ligase buffer | |||
<br>1μl T4 DNA ligase | |||
<br>2μl MQ | |||
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<br> Self ligation: | |||
<br> 8.5μl vector | |||
<br> 2μl T4 DNA ligase buffer | |||
<br> 1μl T4 DNA ligase | |||
<br> 8.5μl MQ | |||
<br> Ligate overnight in the fridge (at 4°C) | |||
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<br> Colony PCR of today was not alright, compare with yesterday's-> yesterday samples should be OK, so plasmid prep and send for <br> sequencing | |||
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Revision as of 06:00, 20 July 2011
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20-7-11
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