daj (David Johnston)
daj at nhm.ac.uk
Fri Aug 5 03:35:25 EST 1994
On Tue, 2 Aug 1994 13:11:34 Pacif,
<dpetersn at unixg.ubc.ca> writes:
>In article <31krfv$8fn at mserv1.dl.ac.uk> (David Johnston) daj <daj at nhm.ac.uk> writes:
>>From: (David Johnston) daj <daj at nhm.ac.uk>
>>Subject: Re: Dissuruption Buffer
>>Date: 2 Aug 1994 08:09:51 +0100
>>On Mon, 1 Aug 1994 18:38:21 GMT,
>> <szcooley at chip.ucdavis.edu> writes:
>>>>Does any one have a recipe for Disruption Buffer used in PCR protocol one
>>>individual E. coli colonies? Thanks,
>>dH2O works fine!
>>(add approx 1mm2 of scraped colony to 40 ul, heat 95C for 5 mins, use 1ul
>>as template in a 10ul PCR reaction (use 10/25th of a standard PCR reaction
>>mix). 5ul of this will give a screamingly bright band on an agarose gel
>>for screening purposes.)
>Heck I just add some scraped cells with a pipet tip to 5 ul H2O and pipet a
>few times to mix. Then, add 20 ul of my PCR master mix, oil, and toss in the
>thermocycler (with a preheat of 5 min at 94C) and all goes smoothly. No
>dilution, no extra steps, just remember to change your tips each time so
>there's no carry-over.
Sure, I agree. It also works if you just scrape the cells into the PCR mix.
I just find that I get cleaner, more consistent results with the extra step.
David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB. England
(tel 071 9389297, fax 071 9388754, email daj at nhm.ac.uk)
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