differential display PCR

Shahram Mori smori at nmsu.edu
Sun Feb 19 22:54:38 EST 1995


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


: 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.

: TIA

: SRLasky

Stephen,
The three most important things 
1) Let the RT reaction go for 1hr.
2) Use random hexamers for cDNA synthesis
3) Up your 10mer concentration to 1uM.

Cheers 
--
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||
88003





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