Reply to Fast& Cheap Plasmid Miniprep Kit

Victor Levenson levenson at uic.edu
Fri May 30 18:36:41 EST 1997


Qguo at aol.com wrote:

>I have used many different commercial kit for Plasmid miniprep, usually
>Quagen's plasmid Purification kit worked well, it worked stable, and fast.
>But recently I am using Omega Biotek's miniprep kit, it works very good, get
>very good yield as Quagen, it is a little bit fast, one miniprep takes only
>10 mins, average yield is about 30mg, the most important reason for me to use
>this kit is that it is much cheap: only $35 for 50 minipreps. I think it is a
>good deal. I hope you call them to asking a free sample or visit their Web
>Page: www. Omegabiotek.com. I hope this information can help you at some
>point. Good luck for your experiment.


This is the post that I recovered from my computer - should have saved
original Mike's massage, but :-(.

Anyway, in his original post Mike states that the method was published
somewhere by somebody, but he does not have the reference handy. I did
not check the original publication either, I guess because the method
worked so well with the first try...

Mike's address is (*@alf1.ngate.uni-regensburg.de). (As I do not know
his last name this is the only way I can acknowledge his
communication)

------------------------------------------------------------------------
------------------------------------------------------------------------

Mike's protocol follows:

* spin down 1.5 ml of o/n culture
* discard sup and resuspend cells in 200 ul of buff A (you can use
Quiagen P1 as well)
* add 400 ul of buff B, shake - slightly - and incubate on ice 5 min
* add 300 ul of buff C , vortex and incubate on ice for 10 min
* spin at full speed 15 min (while spinning prepare new set of tubes
with 500 ul iProp)
* transfer supernatant into new tube with iProp, shake once (or twice)
* spin 15 min
* discard supernatant and wash pellet (almost invisible - be careful)
with 1 ml of abs EtOH
* dry pellets and resuspend in 50 ul H2O or TE.
* use 5 ul for digestion

Buff A: 		50 mM TrisHCl pH 8.0
		10 mM EDTA
		100 ug/ml RNase A

Buff B:		200 mM NaOH
		1% SDS

Buff C:		7.5 M NH4Ac  (NB: this buffer should be prepared every month
since AmAc decomposes - sniff it if you don't believe me, but don't
hold it too close!)
To prepare - dissolve 57.75 g of dry salt in water to make 100 ml.

It goes without saying that lysis buffer also can go bad - NaOH
neutralization by CO2 from air.

=========================================================

This protocol gives consistently good results for minipreps (both RE
digests and sequencing)

Good miniprepping!

Victor Levenson MD/PhD
Res Asst Prof
UIC, Dept of Genetics M/C 669
900 S Ashland Ave
Chicago IL 60607
levenson at uic.edu 
312-413-3887




More information about the Methods mailing list