Corum:PCR

Overview

Standard polymerase chain reaction (PCR) protocol.

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 TAQ DNA polyermerase

Procedure

1. In a PCR tube, mix the components on ice in the order they are listed above.
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.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

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References

Relevant papers and books

1. Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773

Contact

• Sean P Corum
• SC 17:44, 18 June 2012 (EDT):

or instead, discuss this protocol.