Re Testing a Photobiotin labelled DNA probe. Method needed???

Marco Herde mherde at gwdg.de
Fri Feb 4 08:53:31 EST 2005


npercy wrote:
> Thanks for the reply Austin, sorry have to use google groups as the
> newsserver doesn't seem to be functioning at the moment
> 
> You wrote
> 
>>A 500W halogen lamp? Doesn't that give a broad spectrum UV light
> 
> (i.e.
> 
>>maybe you are frying your DNA during photoactivation)? Do you have a 
>>filter on the lamp?
> 
> 
> No filter on the lamp, there wasn't a lot of heat getting into the
> icebucket in which the tube/DNA was contained.  Not sure about the
> liquid temprature but I held my hand there comfortably infront of it. 
> I had a look for what UV ranges the light should give out and it
> should give a full range of visible, some infrared and longer wave UV.
>  I might have a look into trying another light thought not sure where
> I will get it from (maybe use a transilluminator, but I thought that
> would stuff up my DNA)
> 
> 
>>IIRC, most photoactivated groups are activated with long wavelength
> 
> UV
> 
>>(365 nm) and I think that 100W is enough, no?
> 
> 
> The method I have is for a 500W lamp, I've seen 250W used also so
> wasn't sure, these were using photobiotin activation with visible
> light.  Again I'm new to this so I'm still trying to find out about
> it.
> 
> 
>>The other thing to consider is the photostability of the 
>>photobiotin...make sure that it has always been kept in darkness, and
>>that you handle it in the dark as much as you can until after the
> 
> reaction.
> 
> Made up just before christmas, kept in a box, wrapped in Aluminimum
> foil at -20C.  I have minimised the light exposure where I can, so I
> believe that this shouldn't be a problem
> 
> 
>>If you use an anchored avidin/streptavidin (conjugated to acrylate, 
>>sepharose or agarose or the more expensive magnetic support), you can
>>simply bind a fraction of your probe, and then look at the OD260 of
> 
> the
> 
>>supernatant after spinning/filtration/magnetization.
>>
>>Decrease in OD260 will give you an idea of the amount of biotinylated
>>probe versus a control where you don't add photobiotin.
>>
>>The problem is of course that you lose a lot of your probe in the 
>>process, so if you have a small volume spectrophotometer and if you
> 
> use
> 
>>0.2-0.3 OD of material you can limit your loss of material...
>>
>>Cheers
>>
>>Austin
> 
> 
> Yea, thats the problem I see for testing any probes I make, is the
> fact that I could have a lot of loss through the system.  I
> unfortanetly dont have an achored system to test it with.  The thought
> I have been running is the idea of fixing the DNA to something and
> then testing with my Strep-HRP congugate to see if I can get a
> reaction.  The problem with this is its extremely time consuming (a
> day) just to see if the probe has worked, I also dont know whether its
> not working due to other steps either (like the DNA sticking to the
> membrane).
> 
> Thanks for the reply though
> 
> Nigel Percy



More information about the Methods mailing list