FUNGI SILENCING - PROTOCOL TO TRANSFORM FUNGI CELLS (Dec/10/2009 )
HI...I NEED PROTOCOLS TO TRANSFORM FUNGI CELLS...I HAVE A pSILENT VECTOR...BUT I HAVE SOME TROUBLES TO TRANSFORM ESPORES WITH A ELECTROPORATION PROTOCOL....SOMEONE HAVE A DETAIL PROTOCOL FOR TRANSFORMATION FUNGI CELLS?....I NEED TO KNOW SOME DETAILS LIKE IF THE VECTOR WAS LINEAR OR WITHOUT DIGESTION BEFORE THE TRANFORMATION EVENT :S THING LIKE THAT....DETAIL PLEASE
You might try asking your question in the microbiology section also. And please, don't SHOUT :-)
First of all, I transformed sucsessfully two lines with pSilent-1 using protoplast transformation mediated by PEG.
If you're working in a fungal genetic lab, you should have a established procedure of transformation. If not, i advised you to start with something easier that transforming an siRNA vector without homology.
Normally we ecise the product for ko-vectors. In this case you have to linearise the vector backbone and pray. It works for me, but got a huge number of varying phenotypes. If i remeber correct there should be an SpeI site for linearization.
I don't know what you're target gene is, but hopefully something which you can easily detect. (Colour, Fluoresence, Morphology.....)
Recentyl i tried transformation without circular plasmid and got zero transformants.
With best regars