CSPD&background-what membrane?

DAB dboyd at cc.umanitoba.ca
Wed Jun 4 12:25:04 EST 1997


I have been using the Life  Technologies Photogene Detection System for
routine Southern Blots with no problems for years. When I ran out of the
Detection reagent (LumiPhos 530) I found out that I couldn't buy just a
bottle of it from them, I had to buy a whole new kit. So instead I bought
a bottle of ready-to-use CSPD from Tropix and substituted it for the
LumiPhos in the kit as they both can be used for detection using SA-AP
bound to biotin-labelled DNA. 

HOWEVER, though I can see my bands on Southerns I get LOTS of background
now so that my Southerns are not publication quality. I've tried straight
substitution of LumiPhos with CSPD, I've tried using the Photogene Final
Wash followed by incubation with the buffer recommended by Tropix, I've
tried leaving out the Final Wash and just using the Tropix detection
buffer, I've tried blocking the membrane using 5% non-fat milk instead of
3% BSA as described in the Photogene Kit, and I've tried the membrane
recommended for the Kit (Photogene membrane/Biodyne A which is uncharged
nylon) and also some old Hybond Plus (charged nylon). Always lots of
background.

Can anybody recommend a solution? I figure the most likely solutions are
either a different blocking method and/or a different membrane. 

Help, I don't want to bite the bullet and buy a whole new kit from either
company as I have all the components of one if I can just reduce the
background.

Dave Boyd
dboyd at ccu.umanitoba.ca

-- 
Hey, hey, hey, hey! It was the DNA.
Hey, hey, hey, hey! That made me this way.



More information about the Methods mailing list