Sequencing Problem

Solaiman, Dan via methods%40net.bio.net (by Dan.Solaiman from ARS.USDA.GOV)
Tue Oct 16 14:03:07 EST 2007


I would be most interested in learning more about this.  I had past
experience of problem read-through when there is a hairpin structure.
DMSO or betaine did not help me.  I had to manually read the weak
sequencing peaks of the ABI output to barely make out the sequence.  If
other people have better solution I would be grateful to learn more
about it.  

-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu
[mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Sudheendra Rao
N R
Sent: Tuesday, October 16, 2007 8:45 AM
To: Methods from magpie.bio.indiana.edu
Subject: Sequencing Problem

Hi all,
I am using a plasmid vector of BD biosciences to clone ds-siRNA which
has a hairpin loop and  RE overhangs on either end.
to sequence the clone i used Forward and Reverse Primers ordered from a
separate company (desalted and Not HPLC purified) Forward primer
sequence was suggested by BD biosciences company (as in vector spec
sheet) and it is about 60 bp before MCS.
Reverse primer sequence was designed by me and it is 50bp after MCS In
sequencing reaction : Read Length was just 70-90 bp for forward primer
where as for reverse it was 450-550bp. also Quality index and Phred
scores were higher for reverse primer sequences I used another construct
with different siRNA cloned in the vector and it works fine with forward
primer (500-700bp read length) and reverse primer.

My question is: why my first sample did not give good read length for
forward primer where as reverse primer gave. and second sample was okay
with both forward and reverse primers.

I repeated my sequencing twice..and i obtained same results.
Kindly help

Thanks in advance
Sudheendra.


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