dsDNA sequencing protocol

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Tue Nov 29 19:57:34 EST 1994


| Jaz Baker asked for a plasmid sequencing protocol.

If you gopher to net.bio.net, you can find lots and lots of good stuff :-)

For example...<re-post follows>

] From: rybicki at uctvax.uct.ac.za
] Newsgroups: bionet.molbio.methds-reagnts
] Subject: quick n' easy plasmid sequencing
] Message-ID: <201153.292a4f30 at uctvax.uct.ac.za>
] Date: 20 Nov 91 10:04:00 GMT
] 
] I would like to share a quick n'easy plasmid sequencing protocol we have
] recently lifted, which works really well.
] 
] Ku-chuan Hsiao : A fast ands simple procedure for sequencing double stranded
] DNA with Sequenase .  NAR 19:2787
] 
] Basically you simply dissolve clean miniprep plasmid DNA in 25-50ul TE;
] 
] to 5ul add 1ul appropriately diluted sequencing primer (10ng/ul);
] 
] add 1ul 1M NaOH & mix;
] 
] 37 deg C 10'
] 
] add 1ul 1M HCL & mix;
] 
] add 2ul 5X Sequenase buffer
] 
] (optional: 37 deg C 5 min)
] 
] and thereafter go for it according to the Sequenase manual.
] 
] Cuts plasmid sequencing time down drastically, and will really be a help when
] sequencing more than one template.
] 
] Ed Rybicki

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
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