PCR with fluorescein labeled nucleotides

Frederick Garbrecht fgarbrec at lac.jci.tju.edu
Fri Jul 5 20:42:10 EST 1996


rlee wrote:
> 
>      Dear netters,
> 
>      I am interested in doing PCR with a fluorescein labeled nucleotide but
>      am not sure of the potential problems and which base is usually
>      selected to carry the fluorescein.  I would like to hear what all the
>      PCR experts have to say about doing such PCR.  The PCR product will
>      later be used as a probe for hybridization.
> 
>      Thank you in advance.
> 
>      Rozanda
I usually have my oligo synthesized with a 5' fluorescein.  The PCR 
products are then easily detectable using a fluorimager.  5' labelling 
may not give you enough signal for hybridization though; you may need to 
have multiple fluorescein molecules in your oligo for detection, unless 
you are detecting a high copy number target.  For single copy detection 
in southerns, you probably will need to random prime label your PCR 
product with fluoresceinated nucleotide, and even then use a signal 
amplification scheme for detection. 

Good luck,

Fred



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