Corum:PCR: Difference between revisions

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'''Interested in posting a protocol on OpenWetWare?  Here is a template to help you do so.''' 
'''Click the view source tab and copy everything below this line.  Paste it into your new protocol page.  Then replace the text in this page with your own protocol.  Feel free to add or delete sections as appropriate.'''
[[Category:Template]]
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==Overview==
==Overview==
Standard polymerase chain reaction (PCR) protocol for amplification of DNA.


Replace this sentence with a brief description of the protocol and its goal.
==Materials==


==Materials==
For a 50 μL PCR reaction:
List reagents, supplies and equipment necessary to perform the protocol here.  For those materials which have their own OWW pages, link to that page.  Alternatively, links to the suppliers' page on that material are also appropriate.


*supply 1 (i.e. tubes of a certain size? spreaders?)
* 35 μL H<sub>2</sub>O
*reagent 1
* 5 μL 10X PCR buffer
*X &mu;L reagent 2
* 5 μL 2mM dNTPs (each)
**component A (reagent 2 is made up of multiple components)
* 1.5 μL 50mM MgCl<sub>2</sub>
**component B
* 1 μL 50μM sense primer
*equipment 1
* 1 μL 50μM antisense primer
*equipment 2
* 1 μL 5nM DNA template
* 0.5 μL premixed TAQ DNA polyermerase


==Procedure==
==Procedure==
#Step 1
# 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.
#Step 2
# Perform the following thermocycling program:
#*Step 2 has some additional information that goes with it. i.e. Keep at 4&deg;C.
## 95 °C 5 min (initial melting)
#Step 3
## 95 °C 30 s (melting)
##Step 3 has multiple sub-steps within it.
## T<sub>H</sub> 30 s (annealing)
##Enumerate each of those.
## 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 [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==
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'''Relevant papers and books'''
'''Relevant papers and books'''
<!-- If this protocol has papers or books associated with it, list those references here.-->
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<biblio>
#Goldbeter-PNAS-1981 pmid=6947258
#Jacob-JMB-1961 pmid=13718526
#Ptashne-Genetic-Switch isbn=0879697164
</biblio>-->
<!-- Try the [[Template:FormatRef|FormatRef template]]-->
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#{{FormatRef|Goldbeter, A and Koshland, DE|1981| |Proc Natl Acad Sci U S A 78(11)|6840-4| }} PMID 6947258
#{{FormatRef|Sambrook, J and Russell, DW|2001|Molecular Cloning: A Laboratory Manual (Volume II)| | |Cold Spring Harbor Laboratory Press}} ISBN 0879695773
#{{FormatRef|Jacob, F and Monod, J J|1961| |Mol Biol 3(3)|318-56| }} PMID 13718526
#{{FormatRef|Ptashne, M|2004|Genetic Switch: Phage Lambda Revisited| | |Cold Spring Harbor Laboratory Press}} ISBN 0879697164


==Contact==
==Contact==
*Who has experience with this protocol?
* [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]].  
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[[Category:DNA]]
[[Category:DNA]]


[[Category:RNA]]
[[Category:In vitro]]
 
[[Category:Protein]]
 
[[Category:Chemical]]
 
[[Category:Escherichia coli]]


[[Category:Yeast]]
[[Category:PCR]]
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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

  1. 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.
  2. Perform the following thermocycling program:
    1. 95 °C 5 min (initial melting)
    2. 95 °C 30 s (melting)
    3. TH 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.

Tools

Notes

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

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. 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

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

Contact

or instead, discuss this protocol.

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