Hello~ I had some issues with PCR during recent experiments, while some of the sequences having high quality scores, the others are pretty bad. If I re-do PCR on those products, would it be OK to change some of the timing or temperature in my original PCR cycle? I wonder if this could bring bias to my phylogenetic analysis, since the sequences were generated by different cycle settings. Thanks!
Hello Tzon-Han Lin
If you reamplify some of your products, you are essentially purifying them, not changing anything in their sequence — that is, provided that you use a high fidelity DNA polymerase, of course. Thus, it won't affect your phylogenetic analysis.
Hello, Tzon-Han Lin!!
No problem. You will be standardizing your amplification parameters and looking for the purest way to obtain the desired product. Usually, before starting the amplifications, I make a temperature curve with the primer to check the best amplification temperature for my product (looking for it to be pure) using the primer set.
- Badges
- Science method