Alas, poor "magic"! I knew thee well.

Martin Kennedy mkennedy at chmeds.ac.nz
Wed Mar 9 17:34:27 EST 1994


In article <1994Mar2.152928.8680 at mbcf>, neale at mbcf.stjude.org writes:
> I have followed with some amusement the cries of molecular biologists who are
> "lost" without their "magic" plasmid preps. Several authors have quickly
> followed with their home-made versions of the "magic" preps to remedy this
> intolerable situation. 
> 
> Bravo!              
> 
> However, we use a very simple boiling method that processes minipreps in bulk
> using a microcentrifuge. These minipreps are crude but work well for sequencing
> or restriction analysis, and best of all, require no phenol or other "magic"
> reagents. The combination of plasmid and host cell that we use is pBluescript,
> and DH5 alpha or DH10B cells, respectively.
> Geoff Neale

Hear, hear!!  We too use the quickest, crudest miniboils for everything,
including sequencing, subcloning etc.  Provided you use DH5 alpha as the host,
it works.  Other hosts, especially TG1, LE392 etc (the endA1 mutation is
critical).  No phenol, no mucking around, virtually no cost. Using a repetitive
dispensor, polystyrene foam racks that hold the tubes in place when you invert
them over the sink, and a multi-vortexor, making 48 minipreps no hassle, and
you still get time for a morning coffee!

I'll append my protocol after this.
    
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750


	@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
	@                                                         @
	@	Rapid plasmid minipreps by boiling lysis.         @
	@                                                         @
	@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@

References:
Holmes, D.S. and Quigley, M. (1981).  Anal. Biochem. 114, 193-197.


Notes

This procedure yields DNA good enough for double stranded sequencing,
subcloning etc.  It works best for high copy number plasmids, particularly
Bluescript derivatives, and the host strain used is absolutely critical!!! 
DH5a gives good results, but DNA from most other strains will degrade and
cannot be sequenced.  This is due to a salt activated nuclease, endonuclease-1
(DH5a is endA1).  The lysozyme is not important for lysis of cells, it just
seems to help pellet the crud.  It can be left out, but this significantly
reduces yields, and makes sequencing more difficult.  Leaving the preps 24
hours on the bench after making them seems to improve their sequencability.


1. 	Grow 1.5ml O/N in 2xYT.

2.	Pellet cells in 1.5ml Eppendorf tube, then decant broth and vortex
cells to resuspend in residual broth.

3.	Add 200 ul  STET buffer, then 20 ul  of 10mg/ml lysozyme (fresh, or
from frozen aliquots).  No need to mix further.

4.	Pierce the caps with a small needle, and place in a covered boiling
water bath for 90 seconds.

5.	Spin at 12000g for 10 minutes.  During this time, label a new set of
tubes and add 200 ul  of isopropanol.

6.	The pellet of lysed cells varies from being white and compressed, to
being viscous and loose, for no obvious reason - this doesnt seem to affect the
yield of DNA.  Decant the supernatant into the isopropanol tubes, without
trying to the last drop or two.  Mix by inversion or vortexing, then spin again
for 5 minutes.

7.	Decant supernatant, rinse obvious pellet gently with 0.5ml of 70%
ethanol, decant this.  If you arent in a hurry, leave to dry for an hour or so,
otherwise spin briefly and aspirate remaining ethanol.

8.	To the moist pellet, add 100 ul  water containing 1 ul  of boiled 
10mg/ml Rnase A.  With Bluescript, this gives DNA at at least 100 mg /ml.  Use
16 ul  in sequencing reactions, 2 ul  in digestions.



STET buffer:  
				8g of sucrose
				5ml of Triton X100
				10ml of 0.5M EDTA
				5ml of 1M Tris-HCl (pH 8.0)
					
Add millipore water to 100ml, autoclave, and store at RT





More information about the Methods mailing list