sequencing

J. Hendriks hendrikb at worldonline.nl
Mon Jun 29 13:23:50 EST 1998


Hello again
I was asked to give more information. Of course I am willing to give this
information. I do a cycle-sequencing reaction in which I use 33-P endlabeled
primers. The reaction is set on a 6% acrylamidegel afterwards. Normally I
get nice sharp and clear bands, but not the last times.... I have put on gel
several samples: wildtype, parents (I know from my SSCP reaction that the
parent are heterozygote) and a child (homozygote for the mutation). I saw
something on my sequencing-gel I had never seen before. I will try to
explain it. I loadede my gel: all G's, all A's, all T's and all T's. By
doing it like this it is easier to find a mutation. The bands in the upper
and lower layer all look alike and give straight bands. Around the position
of the mutation some strange things are happening: I see double bands. I
know what to expect if a persone has a deletion or a insertion of a
basepair, but that's not the case. I also don't think it's due to the
saltconcentration, because in the other parts of the gel it looks allright.
The distance between two nucleotides (in the gel) is bigger than the
distance between the double bands so I don't think I am looking at real
bands (two single base-pairs). Does anyone have an explanation. It's not due
to the gel, because I have run a other gel with the same result (it was the
same cycle-sequencing reaction).
Please help me out.
Thanks

Brenda
b.hendriks at wkz.azu.nl
hendrikb at worldonline.nl






More information about the Methods mailing list