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Pcr primers - (Feb/06/2014 )

I'm designing primers to simplify a full length cDNA. My issue is the small number of complementary bases!

Forward primer contains 6 extra bases plus restriction site plus Kozak plus 7 complementary bases! This seems to not sound right??!


Dear Jackster, 


I suppose you should have 15-18 bases complementary to ensure that you are amplifying the region you want. 

-Ameya P-

I agree with Ameya P. You do need more bases.


I guess I'll have to do multiple pcr rxns to achieve my goal. My one concern is that I'm amplifying a 7kb cDNA and feel that 3 pcr rxns will increase probability of mutations. Guess I'll need to sequence a lot of clones!


I am completely mystified by  your comment. Your primers should like like this:

5' <18-22 bp matching your target> 3'

What does this have to do with how many PCR reactions you need?


Given the restriction on the Tm being between 65-75 degrees, I can't make my primers very long.- right ? My primers were close to 70 degrees. Btw- my kozak seq was 6 BP.

I have 6 BP extra seq plus 6 BP restriction site plus 6 BP Kozak


You can make your primers as long as you would like. Don't pay attention to the Tm reported by the synthesis companies. The Tm of the 18-22 bases matching your sequence is what matters most. So, the reported Tm will be very much higher than the effective Tm for your PCR reaction.


Thanks guys! Appreciate it! I'll order the new set today.,