Corum:PCR: Difference between revisions
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==Procedure== | ==Procedure== | ||
# In a PCR tube, mix the components on ice in the order they are listed above. | # In a PCR tube, mix the components on ice in the order they are listed above. | ||
# Perform thermocycling program | # Perform the following thermocycling program: | ||
## 95 °C 5 min | ## 95 °C 5 min | ||
## 95 °C 30 s | ## 95 °C 30 s |
Revision as of 14:41, 18 June 2012
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
- In a PCR tube, mix the components on ice in the order they are listed above.
- Perform the following thermocycling program:
- 95 °C 5 min
- 95 °C 30 s
- TH 30 s
- 72 °C 1 min for each 1 kb PCR product
- Repeat steps 2-4 a total of 12-36 times (24 is standard).
- 72 °C 5 min
- 12 °C hold
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
References
Relevant papers and books
- Sambrook, J and Russell, DW (2001) Molecular Cloning: A Laboratory Manual (Volume II) - Cold Spring Harbor Laboratory Press ISBN 0879695773
Contact
- Sean P Corum
or instead, discuss this protocol.