Yeast plasmid miniprep kits?

james_milligan at merck.com james_milligan at merck.com
Thu Nov 2 09:16:23 EST 1995


My apologies for the first post - It got away without the attachment.  
Also sorry about the companies auto generated disclaimer.

I haven't seen commercial yeast mini-prep kits - but this modification 
of Promega's Wizard miniprep kit has worked consistantly well for me.  
My protocol was adapted from an "Promega Notes" article (sorry I lost 
the reference) that they published a year or so ago.  The lysis buffer 
is stable for weeks at room temp. 

Yeast MiniPrep with Wizard Mini-Prep purification
1. Grow 10 mL cultures overnight to stationary phase (selective media 
for plasmid).
2. Harvest cells by centrifugation, decant super, resuspend cells in 
residual liquid and transfer to screw cap microfuge tube.
3.  Pellet cells in microfuge, 20 seconds.  Remove super, resuspend 
cells in 200µl lysis buffer: 

Lysis Buffer
2% Triton X100
1% SDS
100mM NaCl
10mM Tris-Cl, pH 8
1mM EDTA

4.  Add 200µl CHCl3/phenol/isoamyl alcohol, 24:24:1,  0.3 gm acid-washed 
glass beads (0.5mm).
5.  Vortex (high speed) 2 minutes.
6.  Microfuge, 5 minutes.
7.  Transfer super to clean microfuge tube,  add 1 mL Promega Wizard 
mini-prep resin.
8.  Process the resin as per the Wizard kit instructions (Transfer resin 
to mini-column,  apply vacuum to sample,  wash column with wash 
solution, spin put residual liquid from column, elute plasmid with warm 
TE).
9.  Use 5µl of mini-prep to transform 50µl of subcloning efficiency 
E.coli (Gibco's DH5a or HB101) using the procedure provided with the 
cells.




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