PCR-PDF

Overview

Standard polymerase chain reaction (PCR) protocol for amplification of DNA.

Materials

For a 50 μL PCR reaction:

• 35 μL H₂O
• 5 μL 10X PCR buffer
• 5 μL 2mM dNTPs (each)
• 1.5 μL 50mM MgCl₂
• 1 μL 50μM sense primer
• 1 μL 50μM antisense primer
• 1 μL 5nM DNA template
• 0.5 μL premixed TAQ DNA polyermerase

Procedure

1. In a PCR tube, mix the components in the order they are listed above. Keep TAQ DNAP cold right up until it is added to the reaction volume. Mix gently and spin.
2. Perform the following thermocycling program:
   1. 95 °C 5 min (initial melting)
   2. 95 °C 30 s (melting)
   3. T_H 30 s (annealing)
   4. 72 °C 1 min for each 1 kb PCR product (elongation)
   5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
   6. 72 °C 5 min (final elongation)
   7. 12 °C hold (storage)
3. Clean PCR product with PCR Purification Kit.

Tools

• Primer3
• NEB Tm Calculator