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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