sequencing of miniprep dna
David Micklem WCI
drm21 at mole.bio.cam.ac.uk
Fri Jul 15 05:51:44 EST 1994
> ...scads of stuff deleted...
>Melanie - yes! These are the cruddiest, dirtiest preps ever, with big pellets,
>and they sequence beautifully. Instead of going to great lengths adding more
>protein, various detergents etc back into minipreps to get the perfect ds seq
>template, just try the crude boil prep. But remember to use DH5 alpha!!!
>Just harvest cells, resuspend in STET add lysozyme and boil for 90", spin 10
>minutes and tip into an equal vol of isoprop. Spin 5 minutes, rinse in 70%,
>resuspend in water (or TE) add RNase. Use alkaline denaturation, and then
>sequence as normal. 48 preps in a morning is a doddle.
I couldn't agree more! Other endA- strains work just fine too. I've used
XL-1B and BW26 lots of times too. (BW26 is useful 'cos its the strain that
sequencing 'deletion' sets from the Tn1000 (gamma delta) transposon method
end up in). Some endA+ strains DO seem to work at least for some people,
but I'm not sure what they do differently.
There are lots of variants on this method: I add a MIX of STET and a stock of
5mg/ml lysozyme in 50%glycerol - I find this easier than messing around with
the solid the whole time.
If after spinning you pull out the 'snotty' pellet with a toothpick, then add
NH4Ac and Isoprop., the whole procedure only uses one tube, but seems to work
just as well.
Doing lots and lots is especially easy if you have one of those 'grey-rack'
type centrifuges from eppendorf....
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