Standard polymerase chain reaction (PCR) protocol for amplification of DNA.
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
- 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.
- Perform the following thermocycling program:
- 95 °C 5 min (initial melting)
- 95 °C 30 s (melting)
- TM 30 s (annealing)
- 72 °C 1 min for each 1 kb PCR product (elongation)
- Repeat steps 2-4 a total of 12-36 times (24 is standard).
- 72 °C 5 min (final elongation)
- 12 °C hold (storage)
- Clean PCR product with PCR Purification Kit.
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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Relevant papers and books
- Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773
- Sean P Corum
- SC 17:44, 18 June 2012 (EDT):
or instead, discuss this protocol.