Hello and happy monday,
We are about to start screening microsatellites size variation using a
ABI 373 sequencer and the Genescan XL upgrade software. As this is a
first in our lab, I am looking for any advices that could be relevant
to improve our optimization step. The range size of all loci fragments
we are going to survey is believed to be between 100 and 450 bp. We
are going to use both (FAM, TET, HEX and TAMRA (standard size)) and
(NED, FAM, HEX and ROD (standard size)) sets of dyes.
I am mostly interested in advices concerning:
-Importance of playing with gel acrylamide concentration and what
would expect to be the best concentration regarding our fragment size?
-Reaction volume for PCR reactions and dilution of both PCR products
(I guess this should be per loci specific) and standard size marker
(when sequencing, we always use a really lower dRhodamine
concentration than advised by the manufacturer and as far as I
understood the same can be done with the size markers).
-bad and good experience about multiplexing.
-bad and good experience with a given size marker (we have ordered
GS-400HD ROX, GS-500 TAMRA and GS-500 ROX) and with sizing fragments
-Storage of dyes matrix standards, size markers and labelled primers.
For instance, the manufacturer is advising to keep the size marker
between 2 and 8 degrees but I heard that it is however better to keep
them a -20 even for short time storage.
Thank you very much for any help you could provide and I am of course
willing to send back a "summary" to the list if some of you wish to.
Sorry for any crossposting.