Corum:PCR: Difference between revisions
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==Overview== | ==Overview== | ||
Standard polymerase chain reaction (PCR) protocol. | Standard polymerase chain reaction (PCR) protocol for amplification of DNA. | ||
==Materials== | ==Materials== | ||
| Line 13: | Line 13: | ||
* 1 μL 50μM antisense primer | * 1 μL 50μM antisense primer | ||
* 1 μL 5nM DNA template | * 1 μL 5nM DNA template | ||
* 0.5 μL TAQ DNA polyermerase | * 0.5 μL premixed TAQ DNA polyermerase | ||
==Procedure== | ==Procedure== | ||
# In a PCR tube, mix the components | # 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: | # Perform the following thermocycling program: | ||
## 95 °C 5 min | ## 95 °C 5 min (initial melting) | ||
## 95 °C 30 s | ## 95 °C 30 s (melting) | ||
## T<sub>H</sub> 30 s | ## T<sub>H</sub> 30 s (annealing) | ||
## 72 °C 1 min for each 1 kb PCR product | ## 72 °C 1 min for each 1 kb PCR product (elongation) | ||
## Repeat steps 2-4 a total of 12-36 times (24 is standard). | ## Repeat steps 2-4 a total of 12-36 times (24 is standard). | ||
## 72 °C 5 min | ## 72 °C 5 min (final elongation) | ||
## 12 °C hold | ## 12 °C hold (storage) | ||
# Clean PCR product with [http://openwetware.org/index.php?title=Corum:PCR_Purification PCR Purification Kit]. | |||
==Tools== | |||
*[http://frodo.wi.mit.edu/ Primer3] | |||
*[http://www.neb.com/nebecomm/tech_reference/TmCalc/Default.asp#.UBGUE3ikMs0 NEB Tm Calculator] | |||
==Notes== | ==Notes== | ||
| Line 41: | Line 46: | ||
==Contact== | ==Contact== | ||
* Sean P Corum | * [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum] | ||
* '''SC 17:44, 18 June 2012 (EDT)''': | |||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | ||
| Line 47: | Line 53: | ||
<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | <!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. --> | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
[[Category:DNA]] | [[Category:DNA]] | ||
[[Category: | [[Category:In vitro]] | ||
[[Category: | [[Category:PCR]] | ||
Latest revision as of 12:19, 26 July 2012
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
- 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)
- TH 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.
Tools
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
- SC 17:44, 18 June 2012 (EDT):
or instead, discuss this protocol.
