RAPDs

PROBLEM:
I am presently optimizing the RAPD techingue for my research work, and running into a lot of flak. I wish to increase the number of specific amplifications, while reducing the amount of non-specific amplifications. The method I'm currenting trying out involves digesting the DNA prior to the amplification reaction, but non-specific amplification (seemly) create a lot of background smearing that is driving me nuts! What can I do? Suggestions, no matter how trivial would be most appreciated.
This may be a FAQ, but getting answers from the Archive is equally frustrating.
Livinus Emebiri
I use annealing temp of 42!C. A simplistic answer, but it may help. Also, high Mg++ conc can lead to non-specific priming. Choose one primer that gives you a fair number of bands, and run a series to optimize your amplification conditions.
Templates:
1. 25 ng DNA
2. control (water or what ever you DNA is in)
Temperature
1. 32 degrees Celcius
2. 36
3. 40
4. 42
5. 44
Magnesium concentration
1. 1 mM
2. 2 mM
3. 3 mM
4. 4 mM
This will require making 2 x 5 x 4 = 40 reactions, but in one day you can optimize for these important conditions.
e-mail: corley@sfu.ca