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| 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 08:57 PM. |
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| annealing temperature , melting temperature , pcr , protocol , questions |
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