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PCR Protocol Questions

PCR Protocol Questions - PCR - Polymerase Chain Reaction Forum

PCR Protocol Questions - 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 02-21-2011, 09:27 PM
Pipette Filler
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Exclamation PCR Protocol Questions



Hello,

Im designing four different PCR protocol/program for the first time for a cloning project and I could use some help. My issue is with the melting temperatures for the oligos I have; some of them are very high. For one reaction, I have a 78.2* and 92.3* Tm for my primers and another reaction has 92.2*, 88.3* and 78.2* (I am combining sequences in this rxn)

I dont know how to choose a correct annealing temperature for these two reactions. I know the standard way is denature-1' @94*::anneal- 1' @Tm-5*::Extension 3' @72*.

Are these oligos with the really high Tm useful? How can I go about choosing a good annealing temperature? I have only a few weeks left in this project so I cannot start over

Please help! I would love any advice you can give and will answer any questions that I can.

P.S. I should note that the two oligos in the 90s have tails that I am using to attach a new shortish sequence.

Last edited by wanderingstranger; 02-21-2011 at 09:57 PM.
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