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Basic DNA sequencing question


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#1 Sank

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Posted 13 July 2010 - 01:12 PM

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.


#2 bob1

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Posted 13 July 2010 - 05:59 PM

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

#3 Maddie

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Posted 16 July 2010 - 08:21 AM

It can also be due to primer dimers if you had any in your amplification.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#4 entropy2009

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Posted 18 July 2010 - 10:35 AM

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.

#5 Sank

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Posted 21 July 2010 - 11:23 AM

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

#6 Maddie

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Posted 21 July 2010 - 02:49 PM

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.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein




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