help - yeast plasmid rapid miniprep needed!
vvsvetlov at utmem1.utmem.edu
Sun Apr 14 01:26:51 EST 1996
In article <stephan.witte-140496121340 at marvin.biologie.uni-konstanz.de>,
stephan.witte at uni-konstanz.de (Stephan Witte) wrote:
> Hi netters!
> After a two hybrid/interaction
trap screen I have to isolate plasmids
> from my positive clones.
> I didnt succeed with a zymolase protocol, I suppose the enzyme does not
> lyse the cells.
> Are there other reliable protocols, maybe something like alkaline
> lysis...... (not much YEAST experience :-()
> Thanks a lot,
It is not plausible to do anything with a mix of 2 or 3 plasmids (dept. on
reporter being integrated or plasmid-borne) pulled out from yeast cells
directly. After initial specificity tests with positive colonies we grow
them o/night to near saturation in liquid culture (3-5 ml) and then
proceed with rapid-release of plasmid DNA followed by transformation of E.
coli. It (coli) then is minipreped (usually 5 colonies from each
transformation) and bait plasmid is identified by restriction digest (XbaI
for pJG4-5 derivatives). I started using Quiaprep 8 for these minis
because 1/2 miniprep is more than enough for digestion, 2 sequencing
reactions (w/Sequenase), yeast backtransformation and keeping so that I
don't need to midi them. Overall cost seem to be the same (1 midi~5 minis)
but it is much faster. Rapid release protocols are ubiquitous, the one I
use goes like this
RAPID RELEASE OF PLASMID DNA FROM YEAST
1. Pellet cells from 1.5 ml of o/night culture of yeast in Eppendorf
centrifuge. Discard the supernatant.
2. Add 0.2 ml of 2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl
(8.0), 1mM Na-EDTA to the pellet. Add 0.2 ml of phenol: chloroform:
isoamyl alcohol (25:24:1) and 0.3 ml of acid washed glass beads.
3. Vortex at high speed for 2 min.
4. Centrifuge for 5 min.
5. Transfer top layer to a new tube - can be used directly in
transformation of E. coli (2-5 ul) or yeast (15-20 ul), concentrated or
precipitated with isopropanol.
Hope this helps.
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