We have amplified a slightly large segment of K562 cDNA -2271bp- in E.Coli TOP10. At first, we’ve used of MaxTaq Enzyme (Vivantis Com.), but the mutation rate was so great and we found 5 Homozygote mutations. After that, we changed the Enzyme type and we applied the Hi-fid DNA polymerase, as a result, we have identified 3 Homozygote mutations in this stage. It’s interesting that, there were not any shared mutations between these two groups. Before cloning this region as we were not sure about cell line and enzyme fidelity this region was sequenced and we did not face any variation. What’s the problem in your opinion? As you know, we applied several various stages for screening and pureeing the DNA fragment. I don’t know what’s the problem and which step has problem!!
How can I reduce these mutation’s rate? What’s your suggestion?
Use Phusion polymerase or KOD polymerase that are best available high-fidelity enzymes. We have amplified using these polymerases up to 7 Kb with no mutations.
Use Phusion polymerase or KOD polymerase that are best available high-fidelity enzymes. We have amplified using these polymerases up to 7 Kb with no mutations.
I would recommend phusion as well.
In addition you can increase the amount of template DNA, if too little template DNA is added and a mutation occurs in the first couple of cycles the mutation will be propagated to a larger proportion of the final product molecules, howevere with a higher number of initial template molecules there will be a greater number of first round product molecules and with a high-fidelity polymerase its highly unlikely that a mutant product arising later will become a significant proportion of the whole number of amplified molecules. In other words use more template and fewer cycles of amplification.
Please use Q5 (NEB) or Pfu turbo (Agilent) DNA polymerases that normally used for amplifying whole plasmid in site-directed mutagenesis. They are high-fidelity DNA polymerases and therefore you can reduce mutation rates during PCR amplification by using them.
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