Flourescein labeling- Help!

Michele Fuortes mfuortes at med.cornell.edu
Wed Jun 16 18:18:00 EST 1993


Sheila Gay Buzzee writes:

>  Our lab uses a large amount of fluorescein-labeled primers, and we are 
>attempting to synthesize them ourselves instead of remaining at the mercy of 
>suppliers for pure, high-quality primers.  We have no problem making the 
>primers themselves, but we are having problems with labeling.
>
>  Does anyone know how to label the 3' or 5' ends of double-stranded DNA?  (Or 
>both?)  Does anyone have experience with flourescein-labeling primers?  200-bp 
>ds DNA?
>
>  Please send any primary references if possible, or advice if you don't have 
>references.
>

We have been using very succesfully the kit from Amersham to do 3'
labelling. The kit basically includes Terminal Transferase, fluorescinate
ATP and buffers, blocking agents etc. It's a "little" expensive ;-) but it
should not bet too complicated to just get TT and they also sell all the
different fluorescinated NTPs.

Hope this helps.

Michele


----------------------------------
Michele Fuortes, MD
Dept. Cell Biology and Anatomy
Cornell University Medical College
New York, NY
E-Mail: mfuortes at med.cornell.edu



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