Dig detection for Southerns.

[Rod Snowdon]

On Fri, 10 Feb 1995, Duncan Clark wrote:

> I have just bought a DIG detection and labelling kit. Does one have 
> to use Boehringer's own nylon membranes for Southerns or is the 
> background acceptible with others ie HybondN+ or GeneScreen Plus 
> both of which I have. 

I don't find background problems with Hybond N+, despite a Quiagen rep 
telling us that it was "too strongly charged" and we needed to use HIS 
membrane, otherwise we'd be stricken with horrific background and 
jet-black autorads! 


 > Any helpful tips on this kit always appreciated.
       ^^^^^^^^^^^^
Yeah - watch out for errors in Boehringer's (new) instruction booklet for 
their (CSPD) Chemiluminescence detection kit - I've so far found two or 
three excellent ones which could cock things up totally for inexperienced 
users.  Funny thing is their old protocol was OK, but in their nice new 
format they've simplified things to the extent of leaving out some 
slightly-important stuff...

For example:
 
1.  Their "Standard Hyb. Buffer"  when used at the recommended 68 deg. C 
is basically a stripping buffer, since they've omitted the 5*SSC from the 
original recipe.  Nothing will hybridise, including the control reaction...

2.  For reuse of probes they suggest prior redenaturing at 68 degC.  I 
assume this is a typo: 95 degC would be a bit more effective if you want 
to see some hybridisation, depending on how renatured your probes are 
after prior hybridisations...

3.  They forget to remind you to reuse your CSPD solution five times 
before you chuck it out - otherwise you'll run out after ten detections 
(the kit is supposed to last for 50 runs).

And perhaps there's more, who knows???

I must admit to some stupidity here - these discoveries came after blindly 
following the instructions like a cookbook without actually thinking 
about what we were doing until we were faced with blank autorads at the 
end - I guess there's a lesson there for all!!!


Yet to get around to calling Boehringer to abuse them,
Snowy