AFLP rxn signal strength (kinda long)

Mark Berres markb at ravel.zoology.wisc.edu
Thu Aug 31 19:16:46 EST 2000


In article <8oloju$faj$1 at nnrp1.deja.com>,
	kwinkeler at my-deja.com writes:
> I'm currently using the PE kit for my AFLP reactions (dye-primer
> chemistry) and running my reactions on an ABI 377.  However, I'm having
> a problem with low signal strength of my peaks.  I have increased the

You mentioned that increasing the amount of fluorophore labeled
primer only marginally increased the relative intensity of your
fAFLP products. This sounds like a fluorophore degredation
problem. Check to see how old your primers are; certain fluorophores
are more time sensitive than others. Another consideration concerns
whether or not the labeled primers were purified. Even though
most transfer chemistries are 80-90 percent efficient, there still
exists a fair amount of variability. HPLC purify your primers. The
consisteny that results is well worth the extra expenditure of
cash. We use Operon and are very happy with their quality.

Also note that not all fluorophores have identical emission intensities.
You did not mention what fluorophore you were using but
6-FAM gives the greatest signal whereas TAMRA and ROX are much 
weaker. Take a look at my website (URL below) for some more
information. I also have a great solution for 96-well Sephadex-based
purification of fluorophore labeled products.

Finally, I would urge you to make your own reagents, primers etc.
I realize the convenience factor of kits, but doing it yourself
enables much more control of the reaction (or at least the illusion
of control).

				Later,
					Mark
-- 
Mark E. Berres
University of Wisconsin- Madison
http://ravel.zoology.wisc.edu/sgaap
16 T 0303764
     4771858






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