AFLP rxn signal strength (kinda long)

kwinkeler at my-deja.com kwinkeler at my-deja.com
Thu Aug 31 09:03:57 EST 2000


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
amount of labeled primer in the reaction (from the recommended 1 ul
in a 20 ul rxn, to 1.5ul) and increased the number of final PCR cycles
by 5, and although these changes have boosted the signal slightly, it's
not enough.  (I should mention that I'm already loading the maximum
amount of sample possible [1.5ul] on the gel.)  In addition, I've been
trying to concentrate my reactions by using Microcon columns (PE's only
recommendation for concentrating rxns); while these do help, they are
not my "method of choice" for several reasons including variable
recovery volumes and the "pain in the butt" factor when dealing with
many samples at once.

Does anyone have any suggestions for ways to further boost my signal,
either thru manipulating the rxn and PCR conditions, or by concentrating
the reactions?  I've researched many alternatives for concentrating the
reactions, but I can't find anything else that: (1) allows for recovery
in very small volumes (preferably ~ 5ul), (2) will allow me to recover
products as small as 50 bp, (3) is not labor-intensive (96-well format
would be nice), (4) is not super costly (I'd like to come in at <$2 per
reaction for clean-up costs), and/or (5) won't adversely affect the
dye-label (although I admit I'm not real clear on what will or won't
alter the fluorescent dye).  Does anyone know if such a product exists
or know of a company that might be willing to work with me to create
such a product?

Any and all suggestions will be greatly appreciated, and thanks in
advance for your help.

Kim Winkeler


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