WPA Galliformes Genetics Group

given the lack of information on the matter, i hope that folks will post their genotyping problems and solutions (if known) to this board such that we can all help one another troubleshoot genotyping/sequencing problems that arise when gels look like this:

http://gallus.forestry.uga.edu/genotypi … 4_bad.tiff

in this particular case, the 12 in well-to-read gel was run on an ABI 377 under normal run conditions. <s>the polyacrylamide we were using was bad</s>.  polymerization went normally and everything else appeared to go smoothly until the lanes were extracted.  although not from the same gel, once extracted, the size standards in the lane exhibited split peaks as such:

http://gallus.forestry.uga.edu/genotypi … _lane.tiff

<s>as for why the acrylamide went bad, we do not currently know, although we are working on it.</s>

it now appears as if the "blue juice" we were using to help place the comb (96-well) properly in the well (between step-plate and back plate) was causing the problem.  we had switched the blue juice from a formula containing formamide (600ul ddH20, 200ul formamide, 200ul Blue-Dextran - 25mM EDTA solution) to once containing only the Blue Dextran-EDTA mixture and 1X TBE.  upon switching back to the old formula (containing Formamide), the problems appear to have been fixed, and the gel resolved as such:

http://gallus.forestry.uga.edu/genotypi … ygood.tiff

with extracted lanes looking like this, again:

http://gallus.forestry.uga.edu/genotypi … esult.tiff