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PCR with long primers


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3 replies to this topic

#1 thegene

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Posted 26 August 2009 - 01:19 AM

Hi mates,

I have a vector that contains an insert. What i want to do is to make mutagenesis . for that i have designed a sense and antisense primers that is around 52 nucleotides( 15 nucleotides are insert sequence specific and a 35 nucleotides contain a sequence of a binding sites for a bacteriophage coat protein that i want to add to my insert. do any body know or have any idea about the some hints or tips to do such a PCR with a long primers.


Best regards

#2 phage434

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Posted 26 August 2009 - 12:28 PM

There is little special, however I'm concerned that you have only 15 nt overlap of your primer to your template. It sounds like too few, unless there is high GC. You do need to check for primer-dimer and 5' overhang hairpins, which are more likely with long primers.

#3 bob1

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Posted 26 August 2009 - 04:17 PM

The Tm is calculated off the specific sequence, not the whole primer. Use a proofreading polymerase.

#4 thegene

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Posted 27 August 2009 - 06:55 AM

The Tm is calculated off the specific sequence, not the whole primer. Use a proofreading polymerase.



Thanks mates for all your help




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