differential display PCR
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.
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.
Shahram Mori _/\_
Program in Molecular Biology _\ /_
Dept. of chemistry and Biochemistry Box 3C \_ _/
NMSU Las Cruces NM ||
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