PCR: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Barry Canton (talk | contribs) |
Barry Canton (talk | contribs) No edit summary |
||
| Line 2: | Line 2: | ||
== overview == | == overview == | ||
* | * Design primers | ||
* | * Prepare template | ||
* | * Prepare PCR mix | ||
* | * Run PCR cycler program | ||
* | * Analyse by gel electrophoresis | ||
==Designing primers== | ==Designing primers== | ||
Designing suitable primers might be the most crucial step especially | 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== | ||
# 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 | ||
# raise temperature to optimal elongation temperature of | # raise temperature to optimal elongation temperature of [[Taq]] or similar DNA polymerase (72-74°C) | ||
# repeat from top 20-35 times; less cycles gives less product, too many cycles increases fraction of incomplete and erroneous products | # repeat from top 20-35 times; less cycles gives less product, too many cycles increases fraction of incomplete and erroneous products | ||
Revision as of 12:35, 5 December 2006
PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.
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 gneral PCR cycle
- 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
- raise temperature to optimal elongation temperature of Taq or similar DNA polymerase (72-74°C)
- repeat from top 20-35 times; less cycles gives less product, too many cycles increases fraction of incomplete and erroneous products
Specific Protocols
Notes
- A discussion of the amplification efficiencies of different DNA polymerases on templates of varying length and GC content using real-time PCR [1].
References
- Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem. 2003 Oct 15;321(2):226-35. DOI:10.1016/s0003-2697(03)00465-2 |
