Corum:PCR

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'''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.'''
 
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==Overview==
==Overview==
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==Materials==
==Materials==
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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.
 
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*supply 1 (i.e. tubes of a certain size? spreaders?)
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For a 50 μL PCR reaction:
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*reagent 1
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*X &mu;L reagent 2
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* 35 μL H<sub>2</sub>O
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**component A (reagent 2 is made up of multiple components)
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* 5 μL 10X PCR buffer
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**component B
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* 5 μL 2mM dNTPs (each)
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*equipment 1
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* 1.5 μL 50mM MgCl<sub>2</sub>
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*equipment 2
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* 1 μL 50μM sense primer
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* 1 μL 50μM antisense primer
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* 1 μL 5nM DNA template
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* 0.5 μL TAQ DNA polyermerase
==Procedure==
==Procedure==
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#Step 1
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# In a PCR tube, mix the components on ice in the order they are listed above.
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#Step 2
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# Perform thermocycling program
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#*Step 2 has some additional information that goes with it.  i.e. Keep at 4&deg;C.
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## 95 °C 5 min
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#Step 3
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## 95 °C 30 s
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##Step 3 has multiple sub-steps within it.
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## T<sub>H</sub> 30 s
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##Enumerate each of those.
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## 72 °C 1 min for each 1 kb PCR product
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## Repeat steps 2-4 a total of 12-36 times (24 is standard).
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## 72 °C 5 min
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## 12 °C hold
==Notes==
==Notes==

Revision as of 17:29, 18 June 2012

Contents

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

  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform thermocycling program
    1. 95 °C 5 min
    2. 95 °C 30 s
    3. TH 30 s
    4. 72 °C 1 min for each 1 kb PCR product
    5. Repeat steps 2-4 a total of 12-36 times (24 is standard).
    6. 72 °C 5 min
    7. 12 °C hold

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. Goldbeter, A and Koshland, DE (1981) - Proc Natl Acad Sci U S A 78(11) 6840-4 PMID 6947258
  2. Jacob, F and Monod, J J (1961) - Mol Biol 3(3) 318-56 PMID 13718526
  3. 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.


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