---------- Forwarded message ----------
Date: Thu, 2 Jul 1998 11:08:05 +0000 (GMT)
From: 1 <mbad at magnum.barc.ernet.in>
To: "bionet.molbio.methds-reagnts" <bionet-news at dl.ac.uk>
Subject: PCR related problems
Dear fellow news group members.
I am with a group working with a set of microsatellite markers on
HC21 chromosome in Indian population.I have been having problems with
band clarity with locus D21S1910.I tried Jean Weissenbach protocol for PCR
amplification which was 55 deg annealing but there were
non-specific bands.I have increased the annealing temp to 60 deg. this has
given me better results.But certain individual's DNA gives me aound 4
bands at the range of alleles submitted in the GDB.And in some
individuals the bands look like smears around the region.
I have been using 6% PAGE to visualize it.
1) Please advise me on any variations to be carried out on the PCR
protocol to get the specific bands.
2) Do you always get ONLY the clear specific bands with
microsatellites amplification or there are some non-specific bands too.We
have observed few non-specific bands in some of the markers we have been
using.While others are comparatively free of those.
Could you please tell what measures did you take to overcome this.
3) We are into detecting parental origin of extra chromsome in
Down's Syndrome cases.and there is a problem in resolving those bands in
6% ,8% ,10% gels.The bands become diffused in our gels.Do we need to
modify the PAGE conditions,or the % of gel.
I would be thankful if you have tips for these problems.
Thankyou once again.
Suresh Shettigar.
email <mbad at magnum.barc.ernet.in>.
Molecular Biology and Agriculture Division.
BARC,Trombay,
Mumbai-85.
INDIA.
