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Acrobat) This method is often used to prepare miniprep DNA which can be transcribed and translated.
It
takes longer than the alkaline lysis/ammonium acetate method and gives similar quality
DNA but you cannot use alkaline lysis miniprep DNA for in vitro transcription
translation.
Pick colonies to be screened into 2 ml broth containing
appropriate antibiotic. Grow with vigorous shaking at 37ºC
until saturated, typically 8 hrs. Once grown to saturation,
cells can sit around in cold room for days to weeks before
screening, but be sure to label them clearly so you will
remember exactly which set of miniscreens they are!
Chill cells (and while they are chilling put a metal container containing
water to high heat with enough water to cover the bottom 1/3 of
tubes in a rack), transfer to microfuge tube (save residual
cells in tube to be used as starter for large prep of
positives after screening) and spin in microfuge for 20
seconds.
Suck off supernate, resuspend cells by vortex in 200 ul
buffer A, add 10 ul buffer B, mix well and leave on ice for
10 min.
Add 20 ul buffer C, mix well and
Boil exactly one min. We recommend using a metal pan brought to a vigorous boil with enough water to be
able to cover the bottom 1/3 of the tubes. Place a double rack containing samples
only in the lower one in the water -- the upper rack is to keep the lower
rack's tubes from popping -- watching
timer. Using rubber gloves reach in, take out the set of
two racks and as quickly as you can, transfer the tubes to
an ice bath to quick chill. Leave them on ice at least 10
mins then
Spin in microfuge 15 mins. Discard the viscous pellet by
sucking it out carefully with a Pasteur pipet and rubber bulb or using a
yellow tip and a pipettor, being careful
not to lose supernate.
Add 200 ul TE saturated, pH equilibrated
phenol and vortex vigorously. Spin in microfuge 15 secs.
NOTE: PHENOL IS AN ORGANIC ACID AND WILL CAUSE SEVERE BURNS;
YOU MUST WEAR A LAB COAT, EYE PROTECTION, AND GLOVES. READ
THE MSDS SHEET BEFORE HANDLING. YOU SHOULD ALSO WORK IN THE FUMEHOOD.
Remove and discard the lower phase with pipettor or a Pasteur pipet
carefully so as not to lose aqueous phase. As long as you wipe
the outside of the Pasteur pipet tip, and are careful to allow
flow only in one direction, i.e. sucking out of the tube, and
if you wipe off the Pasteur pipet with a fresh kimwipe, you
can reuse the same Pasteur pipet to remove all of the organic
phases. But it is better to learn how to do it with a pipet-person.
Re-extract aqueous phase with chloroform and discard organic
(lower) phase, by transferring the upper phase to a new tube.
Add 20 ul 5 M NH4OAc, vortex, and add 500 ul (2 vol) 100%
ethanol. Mix well and spin down ethanol precipitate, suck off
supernate with a pulled Pasteur pipet and wash with 70%
ethanol (500 ul), repeat spin, suck off carefully and
completely and air dry at 37ºC.
NOTE: ETHANOL IS HIGHLY FLAMMABLE; USE EXTREME CAUTION WHEN
HANDLING LARGE QUANTITIES NEAR A FLAME.
Dissolve pellets in 20 ul TE (no RNase), vortex, spin down for
1 sec in microfuge and they are ready to map either by
restriction endonucleases or transcription-translation and
immunoprecipitation.
Buffer A 50 mM Tris pH 8 / 20 mM EDTA
Buffer B 4 mg/ml lysozyme in 50 mM Tris pH 8 (prepared fresh
daily)
Buffer C 5% Triton X-100 / 40 mM EDTA / 50 mM Tris pH 8
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