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Fast Miniprep Protocol!

Duke Groebe drg at prophet.pharm.pitt.edu
Thu Jul 14 13:51:25 EST 1994


In article <Csxuz7.1Fs at mail.auburn.edu>, johnsbs at mail.auburn.edu (Brandon S
Johnson) wrote:
* 
* I received a ton of mail, so I will post my version of the protocol 
* here.  Please note it is very similar to the protocol published by C. 
* Xiang, H. Wang, P. Shiel, P. Berger and D.J. Guerra.  I had developed 
* this protocol about 1 year ago for my convenience.  Turns out it is very 
* similar to their method.  At any rate, the protocol is....
* 
* 1. Grow cells (I used DH5-alpha) in 4 mls LB Broth with appropriate 
* selective antibiotics overnight (37 C, with shaking).
* 
* 2. Fill Eppendorf tube completely and spin in microfuge about 20 seconds.
* 
* 3. Pour off supernatant and resuspend in 100 microL of GTE (50 mM 
* glucose; 25 mM tris-HCl, pH 8.0; 10 mM EDTA, pH 8.0) with vortex.
* 
* 4. Add 200 microL lysis solution (0.2 M NaOH, 1% SDS) and invert tubes a 
* few times.
* 
* 5. Add 150 microL of 5M potassium acetate and mix by rapid shaking.  Make

* the acetate solution by mixing 60 ml 5M potassium acetate, 11.5 ml 
* glacial acetic acid and 28.5 ml water.  NOTE: This is their recipe, which

* works a little better than what I have used.
* 
* 6. Centrifuge 1 min at high speed.
* 
* 7. With a sterile toothpick, remove the pellet (it should be relatively 
* solid and stable for easy removal).
* 
* 8. Add 1 ml 95% EtOH and invert to mix (I have successfully used both 95%

* and 80%).
* 
* 9. Centrifuge 1 min to pellet, and dry pellet.
* 
* 10. Resuspend in TE (I have used up to 150 microL to resuspend and still 
* obtained a high concentration of plasmid DNA) with 25 microgram/ml 
* RNase.  I have a stock of TE with this concentration of RNase already 
* added that seems to work well.
* 

Hmmm.  I must be getting old.  This is almost identical to an old Maniatis
alkaline lysis ("cold snot") protocol for the preparation of plasmid DNA
from bacteria.  The only significant difference is that the original
phenol/chloroform extraction step has been removed (prior to the EtOH
precipitation).  In addition, I do believe the above "fast" protocol is
identical to the miniprep protocol in "Current Protocols in Molecular
Biology".  

******************************************************************************
The opinions expressed here are my own.  MY very own and NOBODY else's!  I
THOUGHT OF THEM FIRST, HA HA, AND YOU DIDN'T!!  SEE!?  There's my name up
there!  And the date - Ooohh, the DATE!  That's important!  HEE HEE, I'M
GONNA BE RICH!  DO YOU HEAR ME!?  RICH!!  HA HA!!  They're mine...heh, heh,
all mine....  GOD!  I'm so smart!



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