Dig detection for Southerns.
gh66 at mailserv.uni-giessen.de
Mon Feb 27 11:02:56 EST 1995
On Fri, 24 Feb 1995, Paul N Hengen wrote:
> I'm using the Southern-Light [TM] kit from Tropix which also uses the CSPD
> substrate for alkaline phosphatase. For detection, I dilute the stock CSPD
> 1:100 in 0.1 M Diethanolamine, 1 mM MgCl2 pH 10.0. I only use 10 ml at a
> time and then throw it out, but your posting implies I can save it to reuse.
> How do I store this for using it again say, one or two weeks later? Is it
> okay to put it at 4C? Isn't it now contaminated with alkaline phosphatase
> that possibly came off the membrane...and is now in the process of being
> phosphatased to bits?
A phone call with Boehringer assured us that the stuff should be OK
for reuse when stored at 4 deg.C (as for the AMPPD in the original
Boehringer chemiluminesencse kit). So far we've not had problems with
this, though how many times it's reusable is probably pretty dependent on
your probes and how much CSPD signal they "use up" each time. Single
copy DNA probes may last for more than 5 applications (though I've never
tried) and perhaps multilocus or fingerprinting probes may not last for
as many detections. As for contamination by alkaline phosphatase, I
assume the 2*15min Maleic acid washes are good enough to remove that
sufficiently so that it's not a problem(?).
A colleague manages to use less CSPD (to enable him to use it just once) by
"dropletting" 1-2ml (of 1:100 solution in Diethanolamine) along a glass
pipette and rolling that over the membrane, though I prefer the ease of
doing everything in a tube and not having any risk of the filter drying out.
I think I'll continue this way until loss of sensitivity convinces me
that the CSPD can't be reused, but at the mo it seems to be working OK...
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