Pcr primersCloning question
Posted 06 February 2014 - 07:40 PM
Forward primer contains 6 extra bases plus restriction site plus Kozak plus 7 complementary bases! This seems to not sound right??!
Posted 06 February 2014 - 09:56 PM
I suppose you should have 15-18 bases complementary to ensure that you are amplifying the region you want.
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Posted 07 February 2014 - 05:20 PM
I agree with Ameya P. You do need more bases.
Posted 10 February 2014 - 07:05 AM
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!
Posted 10 February 2014 - 08:39 AM
I am completely mystified by your comment. Your primers should like like this:
5' [six bases of trash DNA] [restriction site] [Extras such as Kozak] [18-22 bp matching your target] 3'
What does this have to do with how many PCR reactions you need?
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Posted 13 February 2014 - 07:14 AM
I have 6 BP extra seq plus 6 BP restriction site plus 6 BP Kozak
Posted 13 February 2014 - 07:25 AM
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.
Posted 16 February 2014 - 04:25 PM