Dear all,

I am doing cDNA dot blotting. I am not sure what I need to sufficiently block the membrane if using Church buffer in the hybridisation solution. A freind using denhardts instead of church buffer suggested adding sonicated salmon sperm in the prehyb mix and that this would be sufficient. A lot of Church recepies on the net include BSA is it necessary? As protocols vary so much it seems theres a bit of cooking involved I'm hoping someone can save me a bit of time.

This is the church buffer recipie I am using:
0.5M Phosphate buffer
7%w/v SDS
0.01M EDTA

This recipie has worked well in our lab in the past for other applications


Many thanks

You are wise to compare hybridization to cooking, and a lot of it is trial and error. It may involve a little voodoo as well. Molecular Biologists can be as superstitious as baseball players.

Sheared salmon sperm DNA is a good choice for blocking blots to reduce non-specific binding. BSA helps stabilize the hybridization solution and may also reduce non-specifc binding, especially to the membrane itself.

However, a well designed probe, suitable hybridization conditions and good washing technique can eliminate the need for both. So it kind of depends on your circumstance. If you think your probe may not bind too tightly (because it is short with a low Tm), lower the stringency of your hybridization conditions to ensure binding, then clean it up with step-wise washes, increasing the tempeature and lowering the salt concentration until the background sounds nice (oops, I just assumed your probe is radioactive - I'm old school). If your probe is very sticky or for RNA blots, you may want more stringent hybridization conditions because washing may not eliminate background noise.

Bottom line, begin with the technique (and buffer) with which you are most comfortable - AND for which you have the most friends nearby to help with troubleshooting. Salmon Sperm DNA shouldn't interfere, so there is no harm in adding it. If you have done previous blots without BSA, skip it, you can add it later when you have to repeat it...or, if you have my old boss, do it in triplicate by all possible methods...then still repeat it!

Thanks very much!! I am using radioactivity and a single probe I have designed so this is great advice =)

I used to mess around with denhardts etc but eventually went for a far simpler prehyb / hyb solution:

7% SDS
0.125 M Na2HPO4
0.125 M NaH2PO4
0.25 M NaCl
1 mM EDTA
45% Formamide

pH should be 7.2

I'd never use anything else now.