PCR with fluorescein labeled nucleotides
fgarbrec at lac.jci.tju.edu
Fri Jul 5 20:42:10 EST 1996
> 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.
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.
More information about the Methods