I am struggling to amplify an 7.4 kB fragment which I need to confirm my CRISPR KO. I have tried Pfu and Phusion polymerase, used both HF and GC buffers, tried 50 - 70 C temp gradient, DMSO gradient, used two different templates to make sure my template is clean but nothing works. I used Blast for primer designing and checked the primers against the organism to make sure there are no non-specific binding. i don’t get anything at high temperatures. At lower temperatures there are multiple products including my desired product. But it’s too messy. can anyone help me to solve this ?
It is a hard problem. many tests will be carried out.
1) improve the quality of the extracted DNA.
2) try more polymerase like Q5 DNA polymerase (NEB), PrimeMax (Takara), LA Taq (Takara) and so on.
3) redesign the primer and could extend the primer length up to 35 nucleotides.
Hope you good luck!
Thank you. I will make new primers and look for different Polymerases. Hopefully it will work
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