Differential Display of Artefacts?

Shahram Mori smori at nmsu.edu
Wed Dec 28 19:06:16 EST 1994


Stephen R. Lasky, Ph.D. (Stephen_Lasky at brown.edu) wrote:

: Shahram:  I'm not quite sure how what you describe could work at all.  One
: of the principles of the DD technique is the use of specific 3' primers
: consisting of T12MN (where M is G,A orC  N is G,A,T, or C).  If you use
: random primers for first strand synthesis, you have gotten rid of the
: specificity of the T12xG (A,T, or C) reactions and have to rely on the
: 10mers to prime in both the 5' and 3' directions.  If I'm missing
: something, please let me know.

Stephen, you didn't wrong. As far as I know I am the only one to do this in
a procaryotic system. I use random hexamers for my cDNA synthesis and ONE
( yes one) random 10mer. Initially when I did it it worked for me beautifully.
The gel was ugly so I repeated it again and have been getting mixed results.
In fact it's better for euc's to use random hexamers for cDNA synthesis
as the use of oligodT's gives pretty bad background and false bands (
biotechniques this year). I have been trying to duplicate the procedure.
I checked my RNA and that seems to be good. I don't know if it's my cDNA
synthesis or PCR. The PCR is done at low temps.
I am wondering is the concentration of 10mers is important.

: SRLasky

: -- 
: *********************************************************************
: 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
: ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
: "To me at least, 'Yuck' doesn't capture the full essence of death by
: neurotoxin."   -Dick Dunn
: *********************************************************************
What do you think? It works and then it doesn't work.
Cheers
--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





More information about the Methods mailing list