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PCR protocol questions! - (Feb/21/2011 )


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.


Anything in the 90oC range is too high for the reaction to work, even the 88oC is unlikley. If you have already ordered them just give them a try with a two step PCR. Essentailly you have a 1) denaturing step and 2)combined anneal/extension step (72oC for 1min/kb).

If you haven't ordered yet, I would try and redesign them to a maximum Tm of 78oC. However, note that there are different methods of calculating Tm and they can give very differnt results.

If your primer length (minus the inserted sequences) is around 20mer you could try designing it to 15mer length to reduce temperature. Oh... and I just though of this... I'm not sure if it is correct, but I don't think you include the inserted sequence when you calculate Tm, just the actual primer (someone out there is BioForum land might be able to confirm or refute this comment).

Good luck!


wanderingstranger on Mon Feb 21 21:50:21 2011 said:

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

So you have a normal primer (say 20bp) with sequence added at 5' end, that's not complementary to your target?

In that case calculate Tm for the complementary part only, I would try Primer3+ (Primer_Check) and start the annealing temperature 4 degrees lower than calculated Tm, that works usualy for me.