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