Sequencing Problem

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Wed Oct 17 07:38:26 EST 2007


Dear all,
I appreciate the kind responses.
I would like to bring the question again in forefront..the sequencing did
not specifically work for forward primer and it was fine with reverse
primer..why is that?
i have not used betaine or DMSO but still reverse primer could just read
entire insert including hairpin.
why that?

Thanks in advance
Sudheendra.



On 10/16/07, Sudheendra Rao N R <sudhee26 from gmail.com> wrote:
>
> 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




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


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