Sequencing Problem

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Tue Oct 16 07:45:24 EST 2007


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.


-- 
Think before agree
Think before you nod
but STOP thinking
and You Are God


More information about the Methods mailing list