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Designing primers for cloning

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15 replies to this topic

#16 laurequillo


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Posted 28 October 2009 - 12:08 PM

It's difficult to be sure what stretch constitutes your matching bases (for example, I count 34 bases after your HindIII site in your reverse primer, but you say there are 31 matching bases)...

However, I think you still have way too many, and your Tm's are too high.

You are right, there are 34.

So, still too long. :P

I will follow your advice and go down to 21-25 bases and Tm 60C, and I will do a standard pcr

98 30sec

98 10sec
55 20sec
72 3-4min
35 cycles

72 10min
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