Non-radioactive sequencing

TORRES at EVALUN11.BITNET TORRES at EVALUN11.BITNET
Mon Feb 6 20:49:50 EST 1995


Hi everybody:

    If you will excuse my English, I'd like to ask you a couple of questions.
First, I'm actually testing a method for non-radiactive sequencing based on
DIG-labeled primers. The system is quite simple: the samples run through a
standard acrylamide gel and as they get out from it they are transferred to
a nylon membrane that runs attached to a conveyor belt. Then you only have to
develop the filter using colorimetric or luminiscent detection. So far, the
system has been working quite all right, but I have a (minor) problem as a
result of being a newcomer in this sequencing world. I use a sharktooth comb to
load the samples, but, no matter how thoroughly I rinse the precomb socket, the
samples insist in going into the neighbour wells. The gel is 0.2 mm thick and
the comb too, so I can't understand why. I've been told I need to put some
vaseline in the sharktooth comb, but wouldn't it make a mess of the samples?
Any help would be greatly appreciated.
      And now, if you have stayed with me so far (thanksÙ), my second question:
has anyone tested adding polivinil (sp?) alcohol to buffer 3 in the last
step of the colorimetric detection, that is, in the colour solution? I've heard
it increases the signal in in situs, but I don't know if it's equally efficient
in filter. Somebody out there can help me with this?
      Many thanks in advance from Spain,

                                           Adela



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