Corum:PCR

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Contact)
m (Contact)
Line 47: Line 47:
<!-- 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:PCR]]
+
[[Category:Protocol]]
[[Category:DNA]]
[[Category:DNA]]
 +
 +
[[Category:PCR]]
<!-- Move the relevant categories above this line to tag your protocol with the label
<!-- Move the relevant categories above this line to tag your protocol with the label
 +
[[Category:Protocol]]
[[Category:Protocol]]

Revision as of 17:43, 18 June 2012

Contents

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

  1. In a PCR tube, mix the components on ice in the order they are listed above.
  2. Perform the following 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. 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.

Personal tools