Arnoud van Vliet
avvliet at knoware.nl
Mon Feb 14 19:22:08 EST 2000
Best advice: get yourself another professor, or get her to find the
protocols when she insists you make it yourself.
But kiddin' aside, I would first use the kit, and when that works fine
(which it might not!), then start changing things. Because if you make the
stuff yourself, and it does go wrong, how do you know where the error is.
Most non-RA hybs use antibodies, not biotin. No experience with it myself,
always used the DIG or Gene Images kits.
Maybe best for you would be to do the radioactive hyb: relatively easier and
Tina <buenafesta at yahooNO.SPAMcom> wrote in message
news:38A7EBCC.A6AE5DEF at yahooNO.SPAMcom...
> 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!!
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