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PCR PRIMER DESIGN AND REACTION OPTIMISATION

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trook

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Please find the link for the following method:

PCR PRIMER DESIGN AND REACTION OPTIMISATION
By: Ed Rybicki

http://www.mcb.uct.ac.za/manual/pcroptim.htm

Contents:

Factors Affecting the PCR
Denaturing Temperature and Time
Annealing Temperature and Primer Design
Primer Length
Degenerate Primers
Elongation Temperature and Time
Reaction Buffer
Cycle Number
Nested Primer PCR
Labelling of PCR products with digoxygenin-11-dUTP
Helix Destabilisers / Additives
Useful Universal cDNA PCR Primer
A simple set of rules for primer sequence design

REFERENCES

Compton T (1990). Degenerate primers for DNA amplification. pp. 39-45 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Fuqua SAW, Fitzgerald SD and McGuire WL (1990). A simple polymerase chain reaction method for detection and cloning of low-abundance transcripts. BioTechniques 9 (2):206-211.

Gelfand DH and White TJ (1990). Thermostable DNA polymerases. pp. 129-141 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Innis MA and Gelfand DH (1990). Optimization of PCRs. pp. 3-12 in: PCR Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press, New York.

Krawetz SA, Pon RT and Dixon GH (1989). Increased efficiency of the Taq polymerase catalysed polymerase chain reaction. Nucleic Acids Research 17 (2):819.

Rybicki EP and Hughes FL (1990). Detection and typing of maize streak virus and other distantly related geminiviruses of grasses by polymerase chain reaction amplification of a conserved viral sequence. Journal of General Virology 71:2519-2526.

Rychlik W, Spencer WJ and Rhoads RE (1990). Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18 (21):6409-6412.

Sarkar G, Kapeiner S and Sommer SS (1990). Formaqmide can drrastically increase the specificity of PCR. Nucleic Acids Research 18 (24):7465.

Smith KT, Long CM, Bowman B and Manos MM (1990). Using cosolvents to enhance PCR amplification. Amplifications 9/90 (5):16-17.

Thweatt R, Goldstein S and Reis RJS (1990). A universal primer mixture for sequence determination at the 3' ends of cDNAs. Analytical Biochemistry 190:314-316.

Wu DY, Ugozzoli L, Pal BK, Qian J, Wallace RB (1991). The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction. DNA and Cell Biology 10 (3):233-238.

Yap EPH and McGee JO'D (1991). Short PCR product yields improved by lower denaturation temperatures. Nucleic Acids Research 19 (7):1713.

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 Posted Nov 11, 2006, 21:09 PM
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