- I'm designing primers for Gibson Assembly . My main query about that is the harpain and self dimer formation ? Did you have problems with that ?
Onias Castellanos Collazo from my experience with Gibson Assembly primers, I designed them with the normal 15-25 bp length for the annealing range but then we included a 40-60 bp overhang that is unique to the plasmid backbone that you are inserting the gibson pcr product. So with a long 40-60 bp primer you could get some self annealing but the temperature and annealing we based it off of the 15-25 bp primer length. We had great success with this for over 100+ gibson assemblies.
Thank you so much Shawn!!! Another question is ? Did you have problems with hairpain, self dimer formation ?
Not really at all. When you run it out on a gel you can see diamers at the bottom of the gel but it never interfered with the Gibson assembly. I tested doing a PCR purification afterwards but discovered it really wasn't required as long as you follow the particle ratios listed in the Gibson assembly protocol
These guys designed my Gibson Assembly primers and synthesized 5 ug of the plasmid for me!
https://www.yourbiohelper.com/
They don't have that many templates in their database though...
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