Protoplast procedure? - (Jan/10/2007 )

Hello all

Anybody out there have a good protocol for protoplasting Aspergillus spores? I have tried a number of protocols but what I have identified as my biggest problem is pelleting the protoplasts with centrifugation. They always seen to 'hang' in suspension in a high sorbitol solution. Im just not sure I even have protoplasts left at the transformation stage (a lame microscope). Any help anyone??

Hi Jeff,

I have two protocols that might be useful for you.

Separate the protoplasts from debris by pouring the protoplast/enzyme solution over a miracloth lined filter tube and then place protoplast in a sterile 15 ml Corning tube ( and carefully overlaying the mixture with 10 ml of ST. Spin at 5,000 rpm for 20 min at 4° C in a Keep the protoplasts on ice for remaining of procedure.

Carefully remove the band of protoplasts from the interface of the two solution with a pipette avoid the lower solution. Place the protoplasts in a clean tube tube and add 5 ml of STC.



Spin the pooled protoplasts at 4,000 rpm (4° C) for 10 min. Quickly and carefully pour off the supernatant and gently resuspend the protoplasts in 5 ml of STC.

Alternaria protoplast

To digest the amount of fungus I get from the cultures I use around 7-8 ml
of 10mg/ ml kitalase.

Filter the protoplasts through sterile funnels with sterile
miracloth into 12 ml polypropylene tubes. Wash beakers and funnels with
0.7M NaCl to add to the protoplast.

Centrifuge at 700rcf or a little higher for 10 minutes. Suspend the
protoplasts in 10 ml ice cold sterile 0.7M NaCl by inverting tubes;
tapping is OK but not pipetting. Be patient while you wait for the protoplasts to
resuspend.


Centrifuge at 4º C at 700 rcf for 10 min. Decant the .7M NaCl and resuspend in 10 ml STC. Count protoplast numbers; 5-10million is good for one transformation (more is better).

Centrifuge again at 700 rcf. Decant the STC and resuspend the 5-10 million protoplasts in 70uL of STC. Ten to the 7th protoplasts is what you really want.

i have good protoplasting method of T. reesei, if you are interested ,ask me for this