ReadiLink™ iFluor® 647 FISH Fluorescence Imaging Kit

Before using, thaw all components to room temperature and mix thoroughly by vortexing.

Note: The following protocol can be used as a general guideline to standard DNA FISH. Optimization may be necessary for your experimental system.

1. Prepare the following reaction mixes as indicated in Table 1.

   Table 1. Reagents composition per well for each reaction.

   Components	Volume (25 µL/reaction)	Final Conc.
   FISH Reaction mix (2X)	12.5 µL	1X
   Upstream primer, 10 µM	0.25-2.5 µL	0.1-1.0 µM
   Downstream primer, 10 µM	0.25-2.5 µL	0.1-1.0 µM
   DNA template	1-5 µL	Optimized conc.
   iFluor® 647-dUTP	2.5 µL
   dNTP mix	1 µL
   Water, nuclease-free	25 µL

2. Carefully mix the reagents by gentle vortexing followed by a brief centrifuge.

3. Set up the plate in the qPCR instrument and run as indicated in Table 2.

   Table 2. Thermal cycling parameters.

   Parameter	Polymerase Activation	PCR (30-40 cycles)
   	Hold	Denature	Anneal	Extend
   Temperature	95 °C	95 °C	55-65 °C	68-72 °C
   Time (m:ss)	0:20	0:30	1:00	1:00