differential display PCR

Stephen R. Lasky, Ph.D. Stephen_Lasky at brown.edu
Sat Feb 18 10:12:02 EST 1995

Shahram: In article <3hs2ci$huh at dns1.NMSU.Edu>, you wrote:

> I don't think sending your RNA to be differentially displayed is better than
> doing it yourself. It took me 4 months to make my DDRT work. I didn't any kit
> protocol just reagents. I modified almost EVERY step including the
> concentrations,temperatures and times. 

What were some of your modifications?  About a year ago I started with the
Genehunter kit and then decided that I could do better, so I also modified
concentrations, temps, and times of  everything.  I found that the only
reallllllly critical thing was the quality of your RNA first strand
synthesis.  I found that the cycleDNA first strand synthesis using methy
mercury gave me the best results and the rest didn't matter too much.  

The other thing that we have taken to doing is that if we see bands that
appear different between two samples, we repeat the reactions starting at
the RNA and then run them on another gel.  The trick is to run the gels
for the same time.  Then you can put one gel down on top of the other and
actually confirm whether the first band that changed is the same in the
second experiment.  We find that the major bands on the gels are very
consistent between experiments (which kind of suprised us), but that you
can identify false positives this way and avoid a lot of work.  Then you
can go  on with northerns, slot blots, cloning, sequencing and whatever.

What conditions did you find made a difference?  I am about to have a
student start DD back up and it is always nice to be able to benefit from
others experiences.



Stephen R. Lasky, Ph.D.
Roger Williams Medical Center/Brown University
Phone: 401-456-6572       Fax: 401-456-6569       e-mail: Stephen_Lasky at brown.edu
"The speed of a computer is inversly proportional to the legnth of time it has been on your desk."  Michael Yablonski, circa 1989

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