[Yeast] Yeast spheroplast regeneration

[Wang, Xin-Xiang]

Hello,

I have tried to use sodium alginate method to regenerate yeast
spheroplast.  The methods is as follows:
1. Mix spheroplast with 2% sodium alginate.
2. Layer onto a petridish. 
3. Add a layer of 0.4M CaCl2 to solidify the alginate. 
4. Wash with KCl
5. Add medium to incuate. 

The origninal reference is 
Methods Enzymol. 1993;217:431-6. 
Alginate as matrix support for yeast artificial chromosome cloning.
Cantrell C, Lai E. 

I have not got any colonies from this method.  Has anyone tried this
method?  Anything I am not doing right?  

Also, any other way to regenerate yeast spheroplast?  I need at leat 50%
recovery for my use.  Thanks!

Regards,
Xinxiang Wang