help in doing yeast transformations !!!

Karthi Ramachandran karthi at deakin.edu.au
Mon May 5 15:10:48 EST 1997


In article <Pine.A41.3.95.970504174308.25840D-100000 at aix1.uottawa.ca>, ed <s535290 at aix1.uottawa.ca> wrote:
>
Hi Ed,
I use 20ul sheared salmon sperm DNA @5 ug/ul con as carrier for yeast trans 
and get good eff. To shear the SS DNA, pass the DNA sol. through a 18 gauge 
needle several timed (20 to 25 times)..I dont think you need to check the size 
of the DNA.


For a copy of my protocols, visit my home page
 at www.deakin.edu.au/~karthi/

 and look under Mol Biol Protocols.

Hoe this helps

Karthi. R








>Hi all,
>
>Our electroporator is on the fritz, and I need to do yeast transformations
>preferably using the LiAc method. I have a somewhat modified (and
>much simpler !!!) version from the one that appears on the red book.
>Nonetheless, I read the red book for pointers...the thing that struck me
>was that it mentions that single most critical parameter in transformation
>efficiency when using the LiAc method is the quality of the carrier DNA
>being used...in particular, that it be totally denatured and of a median
>fragment size of about 7 Kb (!?!?!?). The question I ask of anyone who's
>done yeast transformations, is whether you prepare your own carrier DNA or
>purchase it from somewhere ?  It mentions sonication in the shearing of
>the DNA, but without access to a sonicator, I don't know how to go about
>getting the fragment sizes right...in the red book they even suggest
>running an aliquot to check median fragment size...are they being
>unnecessarily anal retentive about this or should I definitely be
>concerned about this ? I don't need an incredible transformation
>efficiency, as long as I have a few different transformants...please share
>your knowledge with me :)
>
>thanks in advance,
>
>Ed
>

Karthi. R
-------------------------------------------------------------------------------
Karthikeyan Ramachandran Ph.D
Research Fellow, Rm. No. SA136
School of Biological and Chemical Sciences
Faculty of Science and Technology	Phone (Off)  61 (3) 5227 1406
Deakin University			Phone (Res) 61 (3) 5241 9806
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Australia 3217				e-mail karthi at deakin.edu.au
Home page				http://www.deakin.edu.au/~karthi/



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