PCR

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
m (The gneral PCR cycle)
Line 11: Line 11:
Designing suitable primers might be the most crucial step in PCR.  This is especially true when using genomic DNA as the template. Traditionally, primers were designed using empirical guidelines. Nowadays, various pieces of software help to predict the best primers including algorithms to prevent mispriming, self-complementarity and primer-primer complementarity, and binding in repeat regions. Additionally, software programs automate the use empirical guidelines for primer design.  See [[Designing primers|here]] for more details...
Designing suitable primers might be the most crucial step in PCR.  This is especially true when using genomic DNA as the template. Traditionally, primers were designed using empirical guidelines. Nowadays, various pieces of software help to predict the best primers including algorithms to prevent mispriming, self-complementarity and primer-primer complementarity, and binding in repeat regions. Additionally, software programs automate the use empirical guidelines for primer design.  See [[Designing primers|here]] for more details...
-
==The gneral PCR cycle==
+
==The general PCR cycle==
# heat template/primer/dNTP/enzyme mix to 95°C for separation of DNA duplexes
# heat template/primer/dNTP/enzyme mix to 95°C for separation of DNA duplexes
# lower the temperature enough for primers to anneal specifically to the template DNA (e.g. 55°C); lowering the temperature too much increases unspecific annealing
# lower the temperature enough for primers to anneal specifically to the template DNA (e.g. 55°C); lowering the temperature too much increases unspecific annealing

Revision as of 12:14, 20 December 2006

PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.

Contents

overview

  • Design primers
  • Prepare template
  • Prepare PCR mix
  • Run PCR cycler program
  • Analyse by gel electrophoresis

Designing primers

Designing suitable primers might be the most crucial step in PCR. This is especially true when using genomic DNA as the template. Traditionally, primers were designed using empirical guidelines. Nowadays, various pieces of software help to predict the best primers including algorithms to prevent mispriming, self-complementarity and primer-primer complementarity, and binding in repeat regions. Additionally, software programs automate the use empirical guidelines for primer design. See here for more details...

The general PCR cycle

  1. heat template/primer/dNTP/enzyme mix to 95°C for separation of DNA duplexes
  2. lower the temperature enough for primers to anneal specifically to the template DNA (e.g. 55°C); lowering the temperature too much increases unspecific annealing
  3. raise temperature to optimal elongation temperature of Taq or similar DNA polymerase (72-74°C)
  4. repeat from top 20-35 times; less cycles gives less product, too many cycles increases fraction of incomplete and erroneous products

Specific Protocols

Notes

  1. A discussion of the amplification efficiencies of different DNA polymerases on templates of varying length and GC content using real-time PCR [1].

References

  1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. . pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]

links

Personal tools