avidin-biotin separation

Basavaraju Shankarappa bsh at MED.PITT.EDU
Tue Aug 31 12:17:10 EST 1993


> Dear netters,
> we are trying to purify some biotinylated DNA fragments with avidin-sepharose
> or strepavidin-sepharose beads. However, since we want to clone this DNA
> we have to detach the avidin from the biotin to recover free DNA molecules.
> The avidin-biotin interactions are extremely strong and we do not know of a
> good means to destroy such interactions. We do not have now the Biotin-UTP
> containing an S-S bridge, which would be easy to cut with a reducing agent.
> So, we wonder if there is anyone there that knows how we could separate 
> free DNA fragments after having recovered the biotinylated DNA. 
> Thanks to all.
> M. Binaschi
> Istituto Nazionale Tumori
> 20133 Milan, Italy
> zunino at mvx36.csata.it
This may be different from the S-S biotin you are referring to. 
There was a very recent advertisement by some company (Pharmacia?)
that a modified version of biotin with significantly reduced affinity
is now available.  This should solve some of your problem.  May be 
someone will remember the exact details.  
	If you cannot change the biotin, I have experienced a reasonable
separation of biotinylated strands from avidin coated magnetic beads by
suspending them in Formamide:EDTA (5:1) and boiling for 2 minutes.  
I cannot assure you that this separation is quantitative, but enough does
come out to be read by an automated sequencer.
Good luck.
Raj Shankarappa
bsh at med.pitt.edu
> 




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