Hello everyone.
It is common knowledge that the chromatograms come cleaner if the PCR products are purified. However, I know some colleagues that send the amplicons to sequence without any process of purification. Usually, they just dilute the PCR product. I myself have done this and I have gotten very clean sequences.
So here is the discussion I want to start. Is amplicon purification necessary? Is dilution a good alternative method for cleaning the DNA? Which dilution do you use to get rid of the primers, Taq, etc.? I know some people only dilute 1/10.
I dilute the PCR product 1/50. The results are usually good, but I sometimes get low quality chromatograms. You can see various examples on the images attached.
I think the biggest problem is carryover of primers from the PCR. Any steps that minimize primer carryover will likely help you to have more reliable sequences. So introducing a spin column or ethanol precipitation of your PCR product before diluting for sequencing might suffice.
I am a fan of the ExoSAPit reagent to clean PCR reactions. It eliminates the unused dNTPs & primers left in the reaction. Looks like you are getting sequence from more than one product in some of your reactions. That's either from actually having a mix of PCR products or from the leftover primers.
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