taq polymerase for sequencing (not cycle sequencing) any luck?

David Johnstondaj daj at nhm.ic.ac.uk
Thu Aug 5 04:23:52 EST 1993


On Tue, 3 Aug 1993 19:58:25 GMT,
  Jon Jafari writes:

>
>Sorry if this is a FAQ, but I couldn't find a FAQ for this group.  
>
>I am having serious proplems with secondary structure and would like
>to know if anyone has used taq to label at a higher temp (or
>sequenase) with success and if so what were the conditions?  I have
>called Promega re their taq kit but they "dogged me like toto" and
>sent the same information that was in their catalog.  Thanks for your
>help.
>
>Jon Jafari

We routinely sequence ribosomal RNA genes cloned into plasmids. Despite 
the potential for secondary structure problems we don't seem to get any. We 
do not use Taq, but Sequenase V2. Follow the protocol as in the Sequenase 
manual but include 10% DMSO in all stages (primer annealing, labelling and 
termination - see Winship, Nuc Acids Res 17, p1266). It helps to start with 
very good quality DNA (eg CsCl-purified or use a good commercial column and 
check purity by spectrophotometer (we use Quiagen)). Keep both labelling 
and termination steps below 5 mins (eg 3 mins each). If you still get 
problems, do the labelling step on ice rather than at room temperature.

Hope this helps,

DAJ
David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB.
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)



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