Chemiluminescent detection

Tina buenafesta at yahooNO.SPAMcom
Mon Feb 14 06:49:32 EST 2000


Hi! I'm planning to use a spot blot for RNA with a DNA probe (labeled
with BrightStar psoralen-biotin kit from Ambion) . My professor insists
that we make the hybridization, wash, and blocking buffers instead of
using their BioDetect BrightStar kit. I'm concerned that the procedure
she wishes to follow (Maniatis) may not be compatible with this setup
because it is intended for radioactive probes. In particular, the
blocking reagent requires sheared salmon DNA and BSA. I found another
protocol in Ausubel specifically for nonradioactive probes on a neutral
membrane. However, the blocking buffer was quite different, requiring a
high concentration of SDS (5%), which I am concerned may distrupt the
positively charged membrane. Furthermore, this buffer did not contain
protein or DNA.
   Should one of these procedure work or if not can someone direct me to
a hybridization protocol compatible with chemiluminescence that doesn't
require buying reagents? Is there a distinct advantage to buying
hybridization buffers and blocking buffers?

I've searched the net, library, and medline without luck.
Any help would be greatly appreciated!!

Tina






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