Hey Arjun,

could you specifiy your question so I get it right. You extracted DNA from an insect, what target do you have? You just wanted to isolate the gDNA or amplify a specific region of interest or the whole gDNA, but after the PCR you have no bands anymore? But positive and negative control did work?

Do you have the opportunity to make a Clean-up after the PCR with magnetic beads to purifiy the samples? Did you measure Nanodrop or Qubit after PCR too?

Hey Hannah,

I am trying to amplify the COX1 gene. Positive and negative controls work. The PCR is failing in insects from two specific collection sites.

I am using Qiagan blood and tissue kit to isolate DNA. Didnt think it was necessary to purify the DNA further. Additionally I extracted DNA from five collection sites simultaneously which included the problematic sites to rule out mistakes during extraction. The same thing was observed as you can see in the gel image. The last two lanes show no bands. I forgot to mention the 260/280 ratio was around 1.8 for all the samples.

Hello Arjun,

If the conservation and transport of the samples was the same and you used the same extraction method, maybe you have some extraction inhibitor in the samples, check how you eluted them and if everything is fine put more cycles in your PCR, they are a few nanograms and if the DNA is a little degraded you will have a small amount, in the end you plant all your sample in electrophoresis or just an aliquot? Have you sequenced the PCR products?

The PCR result is influenced and inhibited by substances used in DNA isolation. You should pay attention to the concentration of the substances you are using and bring them to the appropriate concentration.