Colony lifts - detection with biotinylated probes - background?

[Paul A. Vescio]

In Article <35a9k9$lsj at mozo.cc.purdue.edu>, muriana at aclcb.purdue.edu (Peter
M. Muriana) wrote:
>
>We are using biotinylated probes for detection of target sequences.  
>Currently, we have started using them for detection of clones via
>colony-lifts and have been experiencing background problems with the 
>colonies.  Could this be due to cell-related biotin attracting the avidin-
>alk.phos. conjugate?  Is there a way to get around this problem besides 
>using dig-labelled probes and detection with anti-dig-alk.phos.?  I've 
>noticed that Schleicher & Schuell's colony lift procedure with biotin-
>labelled probes incorporates a step where they hand swab the lifts after 
>they're lysed and baked, and then incubate them in proteinase K solution.
>Just curious if others have experience such background problems, or not.
>Regards, Peter
I had some very good experience with the S&S RADFREE kit for detection.  I
had very little backround and as long as your probe is longer than a 15-20
base oligo you should get good label and very little backround.  I would
definitely recommend using this kit for colony lifts.  And yes wipe after
proteinase K treatment helps alot to reduce backround.  I have also stated
using this wipe step even when not using the S&S kit. 
--------------------------------------------------------------------------------
Paul A. Vescio                      
Biology Dept.
Rensselaer Polytechnic Institute

!!New York Rangers  Stanley Cup Champions 1994!!