Team:Sumbawagen/Notebook/protocol2

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iGEM Sumbawagen 2014 · Econey

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