protoplast preparation and transfection - protoplast! O'God! (Nov/10/2006 )

Hi, all,

Does someone here have good experience with protoplast preparation and transfection? In my case, after transfection most of the protoplast are lyzed and died. I tried different enzyme digestion time course and different enzyme amount. I am a bit crazy with trying and trying and trying, so would someone who has great experience can give some suggestion,

Thanks,

Kuo-Tung.

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It's all about finding a balance between dying cells and poreus cells. What protoplasts are you using? Bacteria, yeast, fungus, something else?
It's better to do a protoplastation for 1 hour with a bit more enzyme, than with less enzyme for many hours. Keep track of them under a microscope, you should see some protoplasts and some "intact" cells. And be very carefull with them.

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Thanks and I am working on the Arabidopsis protoplast.

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I don't have any experience with arabidopsis, so this was all I could tell you. May be you can also open this topic at plant biology

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hi
i have a little experience in protoplast prep.
i used yatalse enzyme sol for 1 hour with gentle shaking at 30c. . i used T. reesei for protoplast prep.

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Thanks all of you, now it works!

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