Corum:PCR

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (Procedure)
Current revision (14:19, 26 July 2012) (view source)
m (Procedure)
 
(One intermediate revision not shown.)
Line 20: Line 20:
## 95 °C 5 min (initial melting)
## 95 °C 5 min (initial melting)
## 95 °C 30 s (melting)
## 95 °C 30 s (melting)
-
## T<sub>M</sub> 30 s (annealing)
+
## T<sub>H</sub> 30 s (annealing)
## 72 °C 1 min for each 1 kb PCR product (elongation)
## 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).
Line 26: Line 26:
## 12 °C hold (storage)
## 12 °C hold (storage)
# Clean PCR product with [http://openwetware.org/index.php?title=Corum:PCR_Purification PCR Purification Kit].
# 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==

Current revision

Contents

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.

Personal tools