PCR Optimization: Reaction Conditions and Components

Consideration for components of the PCR optimization:

Primer Design: Oligonucleotides used for priming the PCR should be at least 16 nucleotides and preferably 18-25 nucleotides in length. They should contain 40% -60% G+C. Avoid sequences which would produce internal secondary structures. The 3’-end of the primers should not be complementary to avoid the production of primer-dimers in the PCR reaction. Avoid three G or C nucleotides in a row near the 3’-end of the primer. Ideally, both primers should anneal at the same temperature, differ not from 5 ?C. The annealing temperature is dependent upon the primer with the lowest melting Tm.

Buffers used for PCR: The standard buffer for PCR contains 50 mM KCl, 10 mM Tris.Cl (pH 8.3 at room temperature), and 1.5mM MgCl2.

Enzyme concentration: 1.25 units of Taq DNA Polymerase. Pipetting errors are the most frequent cause of excessive enzyme levels. The use of reaction master mixes is strongly recommended.

Deoxyribonucleoside triphosphates: dNTPs mix (10mM of each dNTP)

DMSO: About 2.5% can increase product specificity.

Template: 10-100ng for 50 ?l final reaction.

Water: Distilled water which has been filter sterilized or autoclaved.

Last update 29-Oct-2001, Rating of 5 votes.

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	By Satiander Rana on 02-Feb-2009 Dear sir, i mtrying to amplifying cdna and my product is of 2000 bp but i m not able to get any amplification.......my all primer are ok..........please suggest me somthing....... Rating: Reply

	By APIWAN on 20-Sep-2008 Dear sir I'm trying amplify PCR product.Product size is 744 bp but i've got product size about 600 bp. I 've checked my primers. They are all fine. How should I do to get a real product size.? Rating: Reply

	By Kajari Dhar on 21-Sep-2007 PCR product size is 1023. I am getting that product size but I also getting non specific product top of the product. How can I get rid off non specific product? Rating: Reply

	By rakhee lohia on 17-Sep-2007 dear sir i hav one query regarding my pcr. my product size is of 2089 base pairs. i hav checked my primers all r fine but they r not amplifying my product.it is synthesised by diving it in 3 fragments but i want it into a complete fragment. Rating: Reply

	By anubrata das on 27-Jan-2005 this is a great site Rating: Reply

	By lili on 29-Oct-2001 a GC-rich sequence(100bp,GC>90%) with high backgroud smear (using GC-rich PCR system:ROCHE) Rating: Reply

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