I would like to specify my question regarding the transformation of plasmid DNA into F graminearum protoplast. I am using the following reaction mixture (30%PEG 50ul, 200ul protoplast, and 20ug of the plasmid (10ul or 20ul)) after that I put them at room temperature for 20 min and wash out PEG by STC buffer. My question is why shall I use 50ul of PEG? why not the same amount of PEG (220/210ul, depending on protoplast and plasmid amount) as well as Protoplast and plasmid mixture. Secondly, one protocol says 1 hr in ice and then 15 min at RT. What should I follow? However, for arabidopsis transformation, I used the same amount of PEG and protoplast/plasmid mixture,