Dear all, I posted a request on evoldir a while ago trying to find out if anyone had the following problem: Below is the original email: We've encountered a strange problem with fluorescently labeled >> microsat markers in a gastropod - all are amplifying well (and >> produce distinct bands on agarose gels) but when we genotype them on >> our sequencer, we get extremely weak (in most cases unscorable) >> peaks. This problem is taxon-specific: in a fish that we run along >> as a control, we get PCR products with approximately the same >> concentration, but the peaks are easily scorable, so there doesn't >> seem to be anything wrong with our reagents. >> >> Changing the number of cycles (we've tried between 30 and 40) and/or >> salt concentration (2.5 - 6 mM) doesn't solve it. Quite a few people have actually come across this phenomenon, and many said they have been unable to solve it. The most common solution was to use a different species (the problem has been encountered in certain gastropods, ascidians, insects and bryophytes). Below are a number of suggestions [my own comments in square brackets]. Thanks to everyone who responded, Peter 1. Use 1/10 of pre-primer concentration as compared to M13 primer concentration [we were already using 1 uM vs. 5 uM, and diluting it further did not help] 2. Use a different extraction method [we were using either Qiagen kit or a salting out protocol, and salting out was usually the better method. I've now tried CTAB, but that didn't improve genotyping results] 3. Dilute samples 1 in 3 or 1 in 5 [this seems to improve it - particularly diluting 1 in 10 or more te seems to decrease the concentration of whatever inhibitors are in the DNA extractions. The peaks still aren't great, perhaps because DNA concentration also becomes extremely low, but this is really the only modification of the protocol that's improved things a bit] 4. Reduce the concentration of MgCl2 [no improvement, neither was increasing its concentration] 5. Dilute PCR products 1:2, 1:5 or 1:10 prior to adding HiDi [no improvement, in fact, peaks were weaker] 6. Use 2 ul of pooled DNA + 10 ul of HiDi [we were using 1 ul previously, and this doesn't improve it] 7. Boil DNA extractions in Chelex [this seems like a good idea, but I haven't tried it yet. The idea is that boiling destroys the inhibitors and any inhibiting ions stick to the beads] 8. During genotyping, use a lower injection voltage with longer injection time [no improvement] 9. Use different primers [OK, some work better than others, but that's common - replacing all 12 of our species-specific primers is not an option] Dr Peter R. Teske Postdoctoral Researcher Molecular Ecology Lab Dept. of Biological Sciences, E8C Macquarie University Sydney, NSW 2109 Australia Phone: +61 2 9850 8203 Fax: +61 2 9850 8245 E-mail: Peter.Teske@bio.mq.edu.au Website: http://www.bio.mq.edu.au/molecularecology/people.htm Publications: http://www.ru.ac.za/academic/departments/botany_research/peter/ Peter.Teske@bio.mq.edu.au