Hi friends, I am trying to clone some GC rich fragments. The PCRs work well while I use 5% DMSO in the reactions, hardly any band without DMSO. The problem is that the DMSO inclusion largely decreases the PCR fidelity. If only a few kb to be cloned, I can easily sequence a few until I find the right one. I am trying to assemble a few fragments of 10s of kb together, which makes the sequencing screening misserable. Do any of you have suggestion to optimise GC rich PCR with better compromising its fidelity? Thank you. Ziguo
Hi Ziguo
What Polymerase do yo use? Did you think about the standard PCR optimization kits that you can purchase ready or a change to a hot-Start-protocol or a similar change in the thermal settings?
Q5 polymerase will be the best..You dont have to use DMSO and you will have a better result
I very much doubt what your saying is true
- the DMSO will melt secondary structure and render your PCR more efficient but that is quite desperate from PCR fidelity
- PCR fidelity is almost exclusively a function of your polymerase
- WT taq for example has no proof reading capacity or 3’-5’ exonuclease which excises misincorporated based as part of the proof reading principle
- In contrast polymerases like HiFi and phusion are proof readers and thus have the ability to correct mistakes
- make sure you are using a proof reading polymerase and not WT taq (in concert with DMSO)
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