Four-tracking from Hell; (Taq-sequencing?)

Daniel Jacobs d.jacobs at unsw.edu.au
Tue Jul 2 23:34:47 EST 1996


In article <4raijm$oet at epsilon.qmw.ac.uk>, "Dr. A. Ryan"
<a.ryan at mds.qmw.ac.uk> wrote:

> Hi guys,
> 
> I am trying to sequence a GC repeat subcloned into BlueScript but have so 
> far been unable to get past a series of four-tracking (probably due to 
> the high G/C content of the repeat).  I have tried sequencing with 
> deaza-nucleotides and 10% DMSO - no luck.  
> 
> Does anyone have any suggestions on improvements to the standard 
> sequencing reaction or a protocol for sequencing at high temperatures 
> using taq?

Depending on how large your insert is I would try amplifing the the insert
using the universal sequencing primers and deazanucleotides to incorporate
the deazanucleotides into the template. Then try sequencing the PCR
product. I did this once a long time ago (with manual taq sequencing) on a
GC rich template and it worked quite nicely. Hope this is of some help.



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