dsDNA, denaturation, sequencing

rybicki at uctvax.uct.ac.za rybicki at uctvax.uct.ac.za
Tue Jul 21 06:42:55 EST 1992


Completely fool-proof dsDNA denaturing for sequencing:

Ku-chuan Hsiao (1991) NAR 19:2787.

Simply dissolve minprep DNA in 25-50ul TE; take 5ul, add 1 ul seq primer
(10ng/ul); add 1ul 1M NaOH, incub 37degC 10min; add 1ul 1M HCl (incub 10 min if
like); add 2ul Sequenase buffer; incub 37DegC 5 min if like, then follow
Sequenase protocol....  No ppte and desalt and resuspension, cuts max time off
many sequencing reactions, simple enough that honours students take to it like 
canards to HOH. 

Also strongly recommended by the famous Di "deoxy" James, our sequencer par
excellence.
 _________________________________________________________________________
|                                 |                                       |
|  Ed Rybicki                     |    Now you've got the hang of it      |
|  Dept Microbiology              | There's nothing you can't do with it  |
|  University of Cape Town        |         If you're very into it        |
|  PB Rondebosch 7700             |         You can't go wrong....        |
|  South Africa                   |                                       |
|  fax 27 21 650 4023             |              - Mad John               |
|  ed at micro.uct.ac.za             | (Ogden's Nut-Gone Flake, Small Faces) |
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