Hi
so i have before added restriction sites and stop codons by flanking the primer with extra nucleotides. But now the question arises can I add more stuff this way? like a whole His-tag worth of nucleotides? so this would be something in the order of 25 nucleotides?
Should be finde.
I once added 50 nucleotides through a 70 nt long primer.
A small His tag should not be a problem.
The amount of nucleotides that binds to your specific sequence should be ~20 nucleotides in my experience.
The only limit is the length of the primers you use and you can now order those up to 200nts or so. I agree with Sebastian Lühn that you should not have any problems.
Dear Gustav
i'm agree with Sebastian and Michael. i'm generally using PIPE cloning to insert and exchange by pcr the leader sequences for extracellular secretion of mammalian proteins which require the insertion of 60-80 bp and in the most of cases i obtain good results. You need just to work a little in temperature oprimization.
if you are interested to know more about Pipe clonong you can find some video on my blog: ProteoCool https://proteocool.blogspot.com
good luck
Manuele
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