FastPlasmid Mini kit (Eppendorf)
hubahopp at gmx.de
Mon Oct 11 13:31:10 EST 2004
almost all plasmid kits out there work satisfactory. If I have to use them,
I prefer those with ion exchangers. But to tell the truth, I actually hate
them. Following the old Maniatis protocol, one still gets best and reliable
results. but simply for a better price with the same hads-on-time. I even
sometimes use this usually reffered to as "quick but dirty" protocol with
minor modifications for ABI sequencing. In my opinion, mini kits are simply
a waste of money, especially when screening minipreps for the right insert.
If you have some precious samples and want to do high resolution
sequencing, then you may use them, of course, just to be on the good side
of the cake.
Here the protocol comes, assuming you work with standard E.coli that will
1. pellet bacteria in eppi (2 min .lt.5000 rpm in tabletop fuge), drain,
resuspend in 100 ul of 50mM TrisHCl, pH 8, 10mM EDTA or your preferred
similar buffer (non-critical), suspend well eg by infamous scratching
shockwave technology, see below, put in ice for 5 min. If you like, include
some DNAse free RNAse A (0.1mg/ml) (depends on downstream processing, if
too much RNA might annoy your experiment or your supervisor)
2. lyse bacteria with 100 ul of 0.2M NaOH, 1% m/v SDS, never vortex, but
scratch eppi several times across plastic 96 hole eppi holder (or similar
device - that will produce the best shockwaves you'll ever get and ensures
thorough mixing with minimal shearing forces), put on ice again, never lyse
more eppis than 18 or than will fit in your centrifuge as a long incubation
here will kill your plasmids, they won't digest anymore.
3. neutralize with 150ul of 3M potassium acetate pH 5.2 (with cc. HOAc),
add 10ul of tris saturated phenol (that's the extra step that kills "all"
proteins!), scratch again, never vortex. put on ice for 10 min or over
night at -20, then spin (thaw them before if you froze'm to bring forward
step 8a!) for 10 to 15 min at full speed at ROOM temperature (makes nicer
SDS pellet floating on top)
4. transfer supernatant to a new eppi containing 400 ul isopropanol,
avoiding any carryover of white matter (use jellow pipet tips), mix by
shaking for 3.27 seconds, centrifuge for 20 min at full speed.
5. replace isopropanol with 1ml of 70% ethanol (watch the pellet!), invert,
spin for 2 min at full speed
6. replace the 70% ethanol with 100% ethanol, invert and spin again
7. drain the ethanol and dry the pellet, but not too much, actually 15 min
at RT is enough, as it maybe won't redissolvewhen you bake it.
8. redissolve in appropriate volume (say 50 ul) of water or TE and proceede
as usual (digest, pcr, sequence, transfect or whatever you like), if it
doesn't dissolve, store overnight at 4 degC or even RT (but that should
rarely -actually never- happen)
8a. Have coffee / beer / tea
Best of luck and HTH,
At 09:48 11.10.2004 -0700, Brian Greuel wrote:
>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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