A. nidulans Transf. frequencies

Jesus Aguirre jaguirre at IFCSUN1.IFISIOL.UNAM.MX
Tue Sep 19 11:15:53 EST 1995



Dear Colleagues:

Our lab is having trouble with Aspergillus nidulans transformation
frequencies using cosmid DNA to try to clone genes by complementation.
First, we had trouble with Novozyme that seems to be too aggressive.
Things improved after following Howard Brody's suggestion of using Novozyme
at pH 5.0 instead of  5.8.  However, our numbers are too low to think that
we will clone any gene this way.

We are cotransforming a riboB minus strain using 5 micrograms of an argB
genomic library (the argB marker can not be used) and 5 micrograms of
plasmid pPL1 (riboB).  In some cases we are also using 5 micrograms of the
self replicating plasmid pHELP (Clutterbuck). We follow the Yelton et al
protocol (1984).

Numbers are as follow :

argB library plus pPL1                  2 Transformants/100 microliter

of protoplasts.

pPL1 plus pHelp                                 36 Transf.

pPL1 plus pHELP plus argB library               31 Transf.


I remember that Gregory May posted a message saying that CsCl purified DNA
was the best DNA to transform A. nidulans.  I resist myself to believe that
(just because if is true we all will be forced to work much more).  Our
DNAs are purified by Qiagen columns and the DNA we get is great for every
use we have tried (except perhaps for transformation).

I would greatly appreciate your comments and suggestions. Since I feel that
I am not the only one with this problems, perhaps would be better to post
any reply to this message. Otherwise, I promise to edit the answers
directed to me and post them.

Thanks a lot for your attention.

Sincerely,

Jesus.

..........................................................................
Dr. Jesus Aguirre L.
Instituto de Fisiologia Celular-UNAM
Apdo. postal 70-242
04510 Mexico, D. F.
Tel. (525) 622 5651, Fax (525) 622 5630
E-mail: jaguirre at ifcsun1.ifisiol.unam.mx
..........................................................................





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