I have tried a couple of published protoplast/PEG mediated methods and tried a homebrew electroporation (similar to a method which has been very successful for Neurospora), none of which have been successful.
I know that my protoplasting is working as the protoplasts look big and round and recover well before the antibiotic is added. I have a tried and tested positive control for insert DNA so it must be my transformation step that is not working.
I heard from other Botrytis workers that transformation is tricky and needs optimizing, any hot tips would be appreciated.
Different transformation methods are compared in the paper of Ish et al:
http://www.biomedcentral.com/1471-2180/11/266
Here is a pdf of a recent paper in PLoS One that describes Botrytis transformation.
Here is another pdf of a more recent paper in PLoS One that also describes Botrytis transformation.
Thank you for your advice George and Laszlo. I have been attempting to use Antal's method for several months without success until recently. For anybody who stumbles upon this thread with a similar issue, this is how I solved it:
1)I added >50ug DNA, more than I had tried before.
2)Protoplasts are fairly resilient in KC buffer but as soon as you add the PEG solution they burst at the slightest disturbance. Add the PEG extremely gently, no pipetting for mixing, check under the microscope that you still have protoplasts at this stage to determine if you were gentle enough.
I have adapted this method of protoplast fusion / fungal transformation (Please see attached file). It worked really well for me. Please find the paper as an attachment. I hope that will help. Regards
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