generic miniprep protocol

David Adelson davead at prospect.anprod.csiro.au
Sun Feb 27 19:45:13 EST 1994


SIROglopTM SUPERnaturalTM DNA minipreps

February 25, 1994

Robert Seymour & David Adelson  (email to D.Adelson at prospect.anprod.csiro.au)
CSIRO Division of Animal Production
Prospect, NSW
AUSTRALIA



This is a generic version of the Promega Wizard/Magic miniprep
system as tested and implemented in the "PIZZA HUT" at Prospect.
 This method is based on a posting to the Methods & Reagents
news group describing MERLIN minipreps.  We are indebted to the
author of MERLIN and are posting this version in homage not
competition.  You can make a homebuilt vacuum manifold suitable
for this method by capping a 30 cm length of polycarbonate
(perspex) tubing and drilling luer fitting sized holes at ~3 cm
spacing.  Don't forget to make a whole for the vacuum source,
i.e. tap venturi fitted with side arm flask trap.   The nicest
things about this are that you can kiss phenol goodbye and you
can save a lot of money.



1) Reagents



SIROglopTM resin (100mL):

	

	to 66.9g Guanidine HCl add 20mL of 3M Potassium Acetate pH 4.8
	(according to Maniatis, i.e. 3M K, 5M Ac).  pH to ~5.5 with
	Sodium Hydroxide. Heat and stir to bring GuHCl into solution,
	bring to volume of 100mL.  Filter GuHCl and add 10g of Celite
	resin.  Final GuHCl concentration is 7M.



Cell Resuspension Solution:  Use recipe below or use regular
Maniatis Soln.

	50mM	Tris-HCl, pH 7.5  

	10mM	EDTA

	100g/mL 	RNase A



Cell Lysis Solution:

	0.2%		NaOH

	1%		SDS



Neutralising Solution:

	3 M		Potassium Acetate, pH 4.8 (Maniatis)



TE Buffer:

	10mM 	Tris-HCl, pH 7.5

	1mM	 	EDTA



Column Wash Solution:



	200mM	NaCl

	20mM	Tris-HCl, pH 7.5

	5mM		EDTA

	(DILUTE 1:1 WITH 95% ETHANOL FOR USE)



2) Method



-	Grow an overnight culture (~3mL) of the bug of choice.  



-	Make a cleared lysate.



	1.	Pellet 1.5mL of culture in a microfuge (1-2 minutes, top
		speed). 

	2.	Resuspend the cell pellet in 100L of Cell Resuspension
		Solution. 

	3.	Add 100L of Cell Lysis Solution and mix by inverting the
		tube several times.  Continue inverting until the lysate clears
		(very rapid). 

	4.	Add 100L of Neutralising Solution and mix by inversion
		several times.

	5.	Microfuge at top speed for 5 minutes.

	6.	Decant the cleared lysate into a fresh microfuge tube.



-	Vacuum manifold method of purification.



	1.	Add 0.5mL of SIROglopTM resin to the cleared lysate and mix
		by inverting the tube.

	2.	For each miniprep prepare a column.  Take a 1cc disposable
		syringe, using the plunger, force a disk of Whatman 3MM paper
		down to the tip of the syringe barrel. Appropriate disks can be
		punched with a standard paper punch for ring binders.  Insert
		the columns into the vacuum manifold.

	3.	Pipette the the Resin/DNA mix into the column..  Use a
		vacuum to get the resin down into the column. 

	4.	Add 1mL of Column Wash Solution to the column. (This is the
		minimum wash) After the wash has gone through, repeat with one
		or two more wash volumes. (More washes may make it easier to
		digest and/or sequence the DNA).

	5.	Let the resin dry (via vacuum) for a few minutes.  

	6.	When the resin is dry, transfer the column to a conical 15mL
		centrifuge tube and apply 100L of water or TE buffer to the
		column. To increase yields, use water or buffer heated to about
		65-70oC.

	7.	Transfer the tubes containing the syringes/columns to a
		Sorvall GLC or equivalent centrifuge and spin at 2000 RPM at
		room temp for several minutes.

	8.	Remove and discard the syringe/column (they can be washed
		and reused if you are really hard up).  Collect the eluted DNA
		from the centrifuge tube and store as you see fit.  If resin
		fines are apparent in the eluate, microfuge and collect the
		supernatant.  



-	If you don't have a vacuum manifold.



	1.	Perform steps 1-3 of vacuum method.  Once the resin is in
		the column, place the column in a 15mL conical centrifuge tube. 
		Spin the columns in a Sorvall GLC or equivalent centrifuge at
		2000 RPM at  room temperature for a couple of minutes.

	2.	Add 1mL of Column Wash Solution to the column and spin as
		above.  Rewash once or twice more if desired.  (More washes may
		make it easier to digest and/or sequence the DNA).

	3.	Go to step 6 of the vacuum method and proceed from there.



This can be linearly scaled up for 10 mL cultures.  





-	For 100mL cultures:

	1.	Use 10mL of each miniprep solution to obtain a cleared
		lysate.

	2.	Precipitate DNA/protein with 30mL (i.e. equal vol) of
		isopropanol for 30 mins at -20oC and spin at 10,000 x g.  Pour
		off supernatant and save pellet.

	3.	Dry the pellet and resuspend in 2mL TE

	4.	Add 5mL of SIROglopTM resin and use a 10mL syringe as a
		column for the washes (either vacuum or centrifuge).

	5.	Elute with 1-1.5mL or TE or water at 65oC.






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Any opinions expressed herein would probably be regarded as heretical
by the CSIRO and are therefore disclaimed by said organisation.

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