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optimising pcr

optimising pcr - PCR - Polymerase Chain Reaction Forum

optimising pcr - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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Old 02-24-2012, 05:25 PM
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Talking optimising pcr



I had optimised a pcr with a good enzyme, but since it was not very cost-effective I changed over to a cheaper enzyme. Do I have to optimise the protocol again, do a gradient or whatever?

Thoroughly confused.Please help.
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Old 03-06-2012, 02:01 PM
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Default Re: optimising pcr

It depends if you are able to replicate the results obtained earlier using the cheaper polymerase.

If you can't, then yes you will need to optimise all the parameters again.

Keep in mind that it is not a guarantee you will be able to replicate it though. While I haven't had any problems using a cheaper polymerase, some of my friends found that their template or intended region can only be obtained using more expensive polymerases. So it might not end up as the most "cost-effective" option if you spend all the time re-optimising the parameters but are still unable to replicate the results.
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Old 01-31-2013, 05:00 AM
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Default Re: optimising pcr

[Only registered users see links. ] The concentration of each primer should be between 0.1 and 0.5 mM. For most applications 0.2 mM produces satisfactory results. Too high primer concentrations increase the chance of mispriming, which results in nonspecific PCR products. Limiting primer concentrations result in extremely inefficient PCR reactions.
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