Corum:PCR: Difference between revisions
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==Overview== | ==Overview== | ||
Line 20: | Line 4: | ||
==Materials== | ==Materials== | ||
For a 50 μL PCR reaction: | |||
* | * 35 μL H<sub>2</sub>O | ||
** | * 5 μL 10X PCR buffer | ||
** | * 5 μL 2mM dNTPs (each) | ||
* | * 1.5 μL 50mM MgCl<sub>2</sub> | ||
* | * 1 μL 50μM sense primer | ||
* 1 μL 50μM antisense primer | |||
* 1 μL 5nM DNA template | |||
* 0.5 μL TAQ DNA polyermerase | |||
==Procedure== | ==Procedure== | ||
# | # In a PCR tube, mix the components on ice in the order they are listed above. | ||
# | # Perform thermocycling program | ||
# | ## 95 °C 5 min | ||
# | ## 95 °C 30 s | ||
## | ## T<sub>H</sub> 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== | ==Notes== |
Revision as of 14:29, 18 June 2012
Overview
Replace this sentence with a brief description of the protocol and its goal.
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 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
- Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
- Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
- Ptashne, M (2004) Genetic Switch: Phage Lambda Revisited - Cold Spring Harbor Laboratory Press ISBN 0879697164
Contact
- Who has experience with this protocol?
or instead, discuss this protocol.