5 replies to this topic

[Sank]

Hello everyone
I had a basic question about DNA sequencing.
Why is it that the first 15-40 bases of the DNA sequence show poor quality while sequencing?
Even if the base calls are not low, I have always noticed this problem.

Can anyone answer this question.
Thanks in advance

Sankella

Edited by Sankella, 13 July 2010 - 01:13 PM.

[bob1]

IIRC it is due to the difficulties of separating small fragments on the instrumentation used.

[Maddie]

It can also be due to primer dimers if you had any in your amplification.

[entropy2009]

It seems that sequencers cannot yet fix this issue.  The 5' end is always garbled on my sequences, and we use the best sequencer possible right now.

[Sank]

Thanks for the input everyone.

Can this be the reason?
The sequence which is amplified by the primer uses the bases which are labeled by a dye, whereas the primer is not labeled. So may be the sequence of the primer and the initial few bases are not detected by the instrument.

Thanks
Sankella

[Maddie]

Here is what I do to get more data: I use ice and keep all the reagents (and the plate) cold until the moment I put the plate in the thermocycler. It does seem to help.
I also make sure I protect the big dye from light when I use it and when I dry the reactions.