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Spheroplasting and Yeast plasmid isolation

Mr Subhash T subhash at gene.ccmbindia.org
Sat Dec 8 21:30:09 EST 2001

	I am trying to isolate plasmids from yeast two hybrid positives.
The protocol that I am planning to use suggests use of beta glucuronidase
for spheroplasting, folloed by SDS treatment, phenol-chloroform extraction
and ethanol precipitation. The beta glucuronidase that we have is from
Helix pomatia (Sigma). Does this enzyme work on yeast at very low
concentrations? Or rather, I would like to know what is the optimum
concentration of this enzyme that will ensure maximum spheroplast
formation. The protocol says 50 units/0.5 ml culture. But it seems not to
be working. Also, I read about the spheroplasting buffer in other postings
in this group. Is this essential for preparing spheroplasts? Any advice or
suggestion on this matter would be of great help.


Subhash Thalappilly
E-214, Centre for Cellular and Molecular Biology
Uppal Road, Habsiguda
Hyderabad- 500007

Subhash at gene.ccmbindia.org		Phone:91-40-7160222 Ext:1648



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