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extra sequence in primers - (Aug/30/2005 )

I'm going to use the PCR products as a template for in vitro transcription without using plasmids. So I add T7 and SP6 sequences at the 5'-end of my primers (20 bp long, and the primers are also around 20 bp long). Does anybody know if this will have an influence on PCR?
Thank you.



40 bp is fairly large for a primer, but as long as you check for homo/hetero primer dimers and primer hairpins you should be alright.
I don't know exactly how T7 and SP6 interact with their respective promoters, but you should look into it. You may have to add a bit of random sequence upstream of your promoter when you design your primers.

Hope that helps,


Should be alright if they're not complementary to each other. It's fairly long, but for instance if you want to make a primer do PCR and then cut it, you better make your primer really long also and this works so it should be fine.


Do you think there should be extra sequence upstream of the promoter?


Do you think there should be extra sequence upstream of the promoter?

Assuming these are going to be used to transcription by T7 and Sp6 (well not assuming as sherry said they would) I would tack on 4bp upstream of the promoter so the polymerase has a bit more room to sit down on for transcribing. It may have an effect on transcription efficiency, though I have not emperically tested this.