I’m designing a set primer (with melting temp about 60°c) for PCR of a genomic fragment (5 kb). In this region of genomic DNA, there are several very stable secondary structures (about ΔG = -190 kcal/mol at 72°c) that are distributed in 5 kb. Would DMSO addition to this PCR, solve this problem? What concentration of DMSO is needed for this PCR?
DMSO should help. The best solution is using a high fidelity enzyme such Phusion HiFi or Kapa HiFi Hot Start.
Use Phusion® High-Fidelity DNA Polymerase and check Tm in the phusion calculator
I use Phusion High Fidelity to amplify near 5 Kb and work perfectly ...
You can use Q5 polymerase , it has a special buffer for secondary structure formation.
Dear Sameneh.
If you still need to use DMSO if you try at concentrations between 1% to 5% by volume this may help. But, the GC rich kits are the easiest option.
Samaneh Tousi! Can you please mention here that which was best to amplify your desired GC rich gene among all the suggestions which experts left here? You will be highly appreciated as I am also facing the same problem now a days. DMSO didn't work in my case.
Hi Tehmina! Phusion® High-Fidelity DNA Polymerase worked in that PCR, very well. good luck with your work :-)
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