Hello, I am a PhD student interested in population genetics of marine invertebrates. Currently I'm working with a bryozoan: Reteporella.

I have been trying to amplify COI (at least) and, although I finally got some bands, after Sanger sequencing, they turned out to be of bacteria, protists, or crustacea (parasits). I'm working with recent samples (collected between 2018-2019), and also some from 2010 (even got bands in these ones, but no sequence). I have been following the conditions suggested in papers dealing with Reteporella and other bryoans, using Platinum Taq (Thermo Fisher) ou Multiplex PCR Master Mix (Qiagen):

  • Folmer's LCO1490/HCO2198 universal primers
  • 94ºC 3 min; 40x(94ºC 30s, 45ºC 30s, 72ºC 1min); 72ºC 10min
  • 95ºC 5 min, 35x(95ºC 40s, 45ºC 45s, 72ºC 1min); 72ºC 8min

I'm aware these temperatures are quite low and prone to amplify inespecific targets. I'll try a gradient PCR 45-55ºC and a touchdown between the same temperatures.Can someone help me, any tips to help me get specific sequences of Reteporella?

Samples were extracted with PureLink extraction kit (Invitrogen), reccomended for "difficult-samples) after completely crushing the sample with a stainless steel pestle (which is passed on ethanol and fire between samples). Only samples that looked "clean" on the surface with polyps at sight (ensuring they were alive when collected) were extracted.

Thanks in advance!

*Attached are eletrophoreses of the PCRs that worked with annealing temperature of 45ºC using the MM Qiagen or Platinum.

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