I am planning to do Gibson assembly for the very first time. As per the recommendation of the assembly kit, 25-40 bp overlap is required between the plasmid and insert. I have designed a primer pair like 25bp overlap+6bp restriction site+20bp insert sequences. Thus it comprises about 50+ bp.
When I check their oligo properties, they show a secondary structure (more than 3 consecutive bonds, significant negative delG). I tried several tunings but failed to improve the primer dimer formation. Can anybody suggest a better way?
Hello.
It sounds like you're having trouble designing primers for Gibson assembly that don't form primer-dimer interactions. This is a common issue when designing primers with a high GC content or high homology to each other.
There are a few things you can try to improve the primer design:
It's also worth noting that, sometimes primer-dimer interactions can be reduced by adding an appropriate concentration of betaine in the PCR buffer. Betaine is an osmolyte that can interfere with the H-bonds formed between primer-dimer thus reducing their strength.
It's important to test the primer function by performing PCR or running them on gel before Gibson assembly to ensure they're not forming primer dimers or other off target interactions.
In summary, Primer-dimer interactions can be a problem when designing primers for Gibson assembly, but by reducing the GC content, using primer design software, increasing the distance between primer binding sites, adjusting the annealing temperature and introducing mismatches, you can minimize the effect of primer dimers and improve your primer design.
Regards
Did you actually test your primer experimentally? It's totally possible that this will work fine. Not guaranteed obviously. There comes a time where more theory doesn't help more, and I think you're there.
John Schloendorn Thank you for your suggestion that is practically useful.
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