Hybridization background - (Apr/17/2003 )
I need help!
How can I get rid of high hybridization background?
The problem: I did a northernblot hybridization with a 32P random prime labeled 800bp probe. All went fine. After stripping I reprobed the blot with a 32P endlabeled 39mer oligo. But now I'm not able to reduce the background. Any ideas? Has it something to do with the hybridization buffer (containing 50% formamide). Should I increase the concentration of SDS (normally 0.1%)? What is the job of the SDS in the washing solution?
What hybridization buffer are you using? I suggest reducing the concentration of Formamide to 20-30% when using oligo probes. This should allow you to use a hybridization temperature of 42C or higher.
Hope this helps,
Ambion technical support.
I use the following buffer:
5x SSC, 5x Denhardt's, 50% Formamide and 0,5% SDS.
I tested several stripping solutions. Increasing the SDS concentration up to 1%; stripped only with water but the background remains...