international online community for dna barcoding professionals
next is mini-barcode regions if primers can be developed, but that is more costly in both labor and consumables. Most expensive, least tried is to allow the sheared DNA
to be used, but to multiplex on a next gen sequencer - this is one of the true
values of next gen to DNA barcoding - cost a little more, but probable yield is
complete mt genomes, not just COI. Worth a stab and a try if someone can get the $15-$25K to
play with...for a plateful of samples.
Amy Driskell at LAB said:
Lee's right, of course. But we've seen that tweaking PCR conditions (really hammering away at it and trying just about everything) -
types of Taq, additives, concentrations of everything, cycling profiles - can
increase amplification success. Andrea and I have been hitting the old
mosquitoes pretty hard the last few weeks and have seen some improvement. I'd
be happy to share what we've found out. Yvonne Linton apparently has even
better success with old bugs and I hear she has a new PCR technique. She also
generally amplifies the CO1 in 2 or more smaller pieces.
I think, in general, the problem is not small amounts of DNA, but high amounts of PCR inhibitors. This is what the forensics people seem
to believe. But, there's no silver bullet to fix the problem that we've been
able to find.
That's all I know right now. It's very frustrating and success will be expensive in these older specimens.
I am working on Insect barcoding with wide range of order. I am facing problems in amplification of coleoptera and Hemiptera. Could you please help me or suggest me the primer for the order which you have mentioned above?