Is Boehringer Mannheim the only supplier of Dig-labeling

Joseph Polli polli~j at glaxo.com
Tue Jan 24 09:46:41 EST 1995


[This message is converted from WPS-PLUS to ASCII]



In article <93508102105991.511748 at USA> polli~j at glaxo.com ("Joseph 
Polli") write*

>    I have several suggestions depending on what the background
>problems are due to.  If background is all over the filter,
                                        
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
                                          No, it was due to the 
colonies

>  If background is
>due to bacterial colonies, have you tried blocking filters with
>free biotin, then wash, then block with strepavidin, and then
>another wash?  I hope this helps.
>Joe Polli
>Glaxo Research Institute

No, we didn't try that - thanks for the suggestion. Is this 
something that is tried-and-true, or just a thoughtful 
suggestion?  We did try a method suggested by Schleicher & 
Schuell after having seen a biotin-labelled colony lift in their 
advertisement - after colony lifting, lysing, etc., a proteinase 
K incubation of the filters and wiping with Kim-wipes, but it 
didn't work.
Regards, Peter  


Hi Peter,

    The second suggested was just a thoughtful suggestion based 
on previous experience with biotin/avidin detection systems on 
Western blots and tissue sections.  We did not use my second 
suggestion for colony lifts or screen phage libraries.  Our 
protocol sounds very much like what you have tried (i.e. 
proteinase K followed by wiping with Kim-wipes).  Two references 
about the biotinylated probes and methods we used are: Kincaid 
R.L. and Nightiingale, M.S., (1988) Biotechniques 6(1):42-49 and 
Kincaid RL, et. al. (1990) J. Biol. Chem. 265:11312-11319.  I 
hope this will help.  Good Luck.

Joe


Joseph W. Polli
Glaxo Research Institute




More information about the Methods mailing list