Sequencing without precipitation

brett brett at BORCIM.WUSTL.EDU
Wed Apr 23 08:50:13 EST 1997


>I hope that some of you can guide me to a ref/protocol describing how
>to sequence dsDNA without the usual precipitation step following
>denaturation. I'm pretty sure that I've seen such a procedure described,
>but I cant remember where.
>
>If any of you actually use this approach, I would be grateful if you would
>share your protocol and experiences (good or bad) by posting them here.
>
>Thanx in advance!
>
>Troels Wind
>Aarhus University
>Denmark

We have a protocol, loosely based on the Sequenase protocol, which is:
1) 2ul plasmid (1 pmol). Add 0.5ul 1N NaOH. Incubate RT, 5'.
2) Add 6.5ul 1.54x buffer and 1ul (5pmol) of primer.
3) Heat to 70C for 2' and slow cool to 30C. During this time split your
        ddNTP mixes.
4) Make up your labeling mix (per rxn):
        0.5ul alpha 35S-dATP (10Ci/ul, >1000Ci/mmol)
        0.25ul 400mM DTT
        0.2ul dGTP labeling mix (1.5pmol ea. dNTP)
        4.9ul TE (10mM Tris, 0.1mM EDTA)
        0.2ul enzyme
5) Add 6ul labeling cocktail to each template/primer mix and proceed.

1.54x buffer:
        0.615ml 1M Tris, pH7.5
        0.154ml 1M MgCl2
        1.0ml   0.77M HCl
        8.23ml  H2O

Hope this helps


Brett Lindenbach
    
Program in Immunology                              
Washington University - St Louis                  
brett at borcim.wustl.edu                             




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