AFLP rxn signal strength (kinda long)

Mr JN Goulding jgouldin at hgmp.mrc.ac.uk
Thu Aug 31 09:53:17 EST 2000


I used to do a lot of FAFLP and started off using the kit, but got very
poor results. In the end I ordered my own labelled primers and didn't use
any of the kit reagents, bought everything in myself. A 20 ul reaction
gave very good results- though I did have a slight problem with the colour
overlap when multiplexing. If only using one labelled primer then FAM
label it (blue) which didn't give any overlap with the ROX labelled
marker. If you need more details than that (such as who we got reagents
off) then feel free to email me and I'll send you the details (I don't
have them at hand) and the refs for the papers. have fun,

Jon

On Thu, 31 Aug 2000 kwinkeler at my-deja.com wrote:

> 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
> 
> 
> Sent via Deja.com http://www.deja.com/
> Before you buy.
> 






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