Hybridization problems - Need advices (Apr/10/2005 )

Hi,

I am doing reverse hybridization. I labeled the probes onto the Hybond N+ membrane using alkaline fixation. After that, the membrane will go through pre/hybridization using Church Buffer, followed by washing. I used 5%BSA in Tris buffered saline-Tween to block the membrane. Then the membrane was incubated in Streptavidin-AP conjugates in TBS buffer. Finally, NBT/BCIP was used for precipitation.

My problem is i cant get any positive signal from the membrane using the mentioned protocols. The background existed. First i suspected there is a problem for my probe labeling onto the membrane. Any suggestion of fixing the probes onto the Hybond N+ membrane?? Do i need to bake the probes-fixed membrane??

Secondly, i suspected it may due to the concentration of my probes and the biotinylated PCR products. Any advise for these concentration??

Please advise.

Thank you.

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Hi,

Anyone here can give some hints to solve my problem??

Thanks.

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could be that you need to work with 5' attachment of probe?

could you send me also what others think, I am facing similar problem.
ibrukner@hotmail.com

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I still cannot obtain any positive signal from the experiment. But i got some improvement. The labeling of the probes onto the membrane should be alright. I used the same way (alkaline fixation) to label my biotinylated PCR products onto the membrane. After the washing, streptavidin-AP incubation, and NBT/BCIP precipitation, the expected signal was obtained.

Right now, i suspect the problem is came from the hybridization step. May be the biotinylated PCR products concentration, the buffer, or the duration of hybridization.

Anyone has experience this?? Please advise.

Thank you.,

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I son't know if this is your case, but myself and several other labs here at MIT have had problems with alkaline trasnfer onto Hybond N+. It seems Amersham is aware of the problem that could be due a change in manufacturing. In my experience the transnferred DNA does not covalently bind to the membrane and is largely washed away during or after Hybridization.
We solved the problem, after many months wasted, by doing high salt transfer (20XSSC0 followed by UV cross-linking using an old batch of Hybond N.

Good luck.

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