I want to amplify a cDNA (1600bp) using Taq polymerase which I have amplified using Pfu polymerase. I have tried all changes I can do like temperature variation, increased MgSO4 concentration and even the buffer exchange with HF buffer, but the result was negative all the time with Taq. Can anyone suggest what could be the probable reason for this failure?
Pfu is a different version of the DNA polymerase. It's sometimes the only choice that works with "difficult" templates (high self-annealing, repetitive sequence, etc.). Sometimes the only way to proceed with your product is to just use what works and call it done.
Which brand of Taq are you using? Promega's Go Taq Flexy is usually nearly as robust as Pfu, but if the fragment have high complexity (repetitive, GC Rich) could still be challenging. If the problem is GC content increasing denaturing step or adding DMSO (5%) or Betaine (around 3%) could help you.
@Roser Thank you for the suggestion. I used 1.5 M Betaine and it worked with Taq DNA polymerase (NEB).
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