cycle sequencing with 35S??

John Nash nash at biologysx.lan.nrc.ca
Wed May 19 18:59:15 EST 1993


In article <erman-190593172439 at 129.64.1.96> erman at brandeis.edu.bitnet (batu erman) writes:
>From: erman at brandeis.edu.bitnet (batu erman)
>Subject: Re: cycle sequencing with 35S??
>Date: Wed, 19 May 1993 21:28:16 GMT

>In article <C7AB34.35M at ccu.umanitoba.ca>, jrichmn at ccu.umanitoba.ca (Joy
>Richman) wrote:
>> 
>> Dear Netters,
>> 
>> Has anyone out there got cycle sequencing to work using 35 S? If so I
>> would like to know what kit you used and how much dsDNA you started with.
>> I would like to get away from using 32 P since we are not doing it often
>> enough to get through even the smallest amount of gamma 32 P before it
>> expires.
>> 
>> P.S. We have the Gibco kit but they do not recommend its use with 35 S.
>> 
>> -- 
>> The most important event in life is not birth or death but gastrulation.
>> Lewis Wolpert
>> jrichmn at ccu.umanitoba.ca
>Promega also says one should kinase the primers instead of incorporating 
>the 35-S or 32-P into the extension products. Why is this? I cannot think
>of 
>any good reason. 
>Batu Erman

'twasn't a kit... I made up the components, but NEB's Circumvent cycle 
sequencing system, which uses Vent polymerase instead of Taq pol, works fine 
with 35-S.  Yes, there is also a kit available, (and I even used it once 
<blush>, and liked it).  I used the incorporation method, as opposed to the 
kinased-primer method.



cheers, John

John Nash                           | Email: Nash at biologysx.lan.nrc.ca.
Institute for Biological Sciences,  | National Research Council of Canada,
Cell Physiology Group.              | Ottawa, Ontario, Canada.
             *** Disclaimer:  All opinions are mine, not NRC's! ***



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