FastPlasmid Mini kit (Eppendorf)

Christiaan Karreman karreman at onkochemie.uni-duesseldorf.de
Tue Oct 12 02:18:10 EST 2004


here is a one tube mini-prep method, also referred to as: the fast
boiling method.


- grow your E.coli in LB or TB with antibiotics
- spin your cells for 15 secs in eppi
- discard the supernatant and
- suspend your cells in 350 ul STET
     STET:
           10 ml Triton X100
           20 ml EDTA 0.5 M
           10 ml Tris pH 8.0 1.0 M
           16 g Sucrose
           add water to 200 ml

- you can now add lysozyme (2 ul 10 mg/ml)
   but it is not really necessary for most E. coli
- 60 secs 100 degrees (really 100, BOILING waterbath)
- spin it down (say 20 min in eppendorf or 10 min in SA600 rotor 40,000
g)
- take the pellet out with a toothpick and throw it away, leave the
supernatant in the eppi
- add 350 ul isopropanol to the supernatant, mix it well
- 15 min in eppendorf centrifuge, full power
- discard the supernatant, dry the pellet
- solve the pellet in 100 ul TE
- use 10 for digest


Christiaan




Brian Greuel schrieb:

> Have any of you tried the FastPlasmid Mini kit offered by Eppendorf
> for preparing plasmid minipreps?  It purports to utilize a single
> solution to resuspend, lyse, and neutralize bacterial cells, making
> the process of preparing plasmid minipreps considerably faster.  An
> Application Note published in the September 2004 issue of American
> Biotechnology Laboratory describes this product.  According to the
> article, the quality of the plasmids produced with this kit is high
> enough to get excellent results with restriction digests and cycle
> sequencing.  Just a disclaimer -- I have no connection with this
> company whatsoever.  I'm simply inquiring as to whether people's
> experience with this product lives up to the claims.
>
> On a related note, does anyone have any tried and true recipes for
> home-made solutions used in a single-step plasmid prep protocol?




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