Success with Multiplex

After a month of PCR trouble shooting I’ve finally worked out a methodology that will work consistently for my specimens!  DNA purification, as I have mentioned previously, worked inconsistently so I turned to PCR additives and Qiagen Multiplex Taq . Following by the protocol developed by Thormann et al (a great paper by the way – see below), I initially had success with my staphylinids  (including Atheta TFIC sp 01 below) using the 28S D3 marker. No surprises there as this was what is the primer set was designed for. However I was surprised by its consistency – every specimen worked perfectly even if the DNA template wasn’t good. I was possibly a little too excited to see nice clean bands on the gel! I also tried the same protocol without Q solution and it didn’t work – just showing how important PCR additives can be.

Buoyed by success I tried a mixture of beetle families, and a again success. Then I tried COI Folmer primers  with a lower annealing temperature- again success! Thormann et al’s protocol seems to be quite flexible and deals with pitfall trapped beetles of many family’s well.

Now we will see if it works for other markers…..

(http://www.dnabarcodes2009.org/meeting_documents/Wednesday/Session%20D/Auditorium%20B%20-%20Wednesday%20-%20Thormann.pdf)

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