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Team:Sumbawagen/Notebook/protocol2
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Latest revision as of 09:11, 19 February 2015
Team:Sumbawagen/Team
From 2014.igem.org
Notebook – Protocol – 2.PCR (Polymerase Chain Reaction)
1. Ordered primers were dissolved in 1x TE buffer to get a 100 uM stock, then diluted to 10 uM stock by 1x TE buffer (usually to final volume of 200 ul). Primers stocks were stored at -20 °C.
2. There were 3 pairs of primers for the PCR namely
- ACEF/ACER1 to clone catalytic domain sequence of adenylate cyclase gene
- AEF/AER to clone full length sequence of IIA(Glc) gene
3. Reagents compositions for PCR were as follow:
- 2X PCR master mix (Thermoscientific), 25 ul
- Sterilized distilled water (Akuabides), 15 ul
- 10 uM forward primer, 5 ul
- 10 uM reverse primer, 5 ul
- Sterilized distilled water (Akuabides), 15 ul
- 10 uM forward primer, 5 ul
- DNA template, colony from agar plate 0 ul Total volume, 50 ul
4.Reaction conditions for PCR were as follow:
- 95 °C, 2 minutes
- 95 °C, 30 seconds
- 53 °C, 30 seconds
- 72 °C, 2 minutes
- 72 °C, 7 minutes Total cycle, 35 cycles
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