magnetic bead losses during manipulation

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Thu Jan 25 12:22:18 EST 1996


W.Coco (ifai at po.uni-stuttgart.de) wrote:

: Thanks for the tips, Paul. I don't have an explanation for the binding of
: BSA to beads. Does the BSA that sticks interfere with your experiments;
: including after its elution upon boiling? I could imagine it might even
: be helpful as a blocking agent to prevent the non-specific binding of
: unwanted and uncharacterized proteins or the adsorption of rare DNA-
: binding proteins one might try to capture with biotin labeled DNA. Of
: course I can also imagine where it might bother (e.g. if it has the same
: migration as a target protein).

Well, for DNA binding protein studies, the negative control always has a
band. Then when you don't see the other expected band, you begin to worry
that the BSA was blocking it somehow. I've decided to leave BSA out.

: Paul, you mentioned how you siliconize with Sigmacote, but didn't indicate
: a washing step (as does Maniatis for dichlorodimethylsilane). Is this
: not necessary only with your method or would you also not rinse after
: Maniatis' method (p. E.1 in the 2nd ed.)? If not, the Maniatis method,
: performed in a vacuum desicator, allows
: one to treat lots at once (of course they warn you could destroy your
: vacuum pump if not careful).

I've never done it the Sam/Maniatis way. As with many of their protocols,
I find it much too complicated. For example, they still tell you to extract
your phenol with TE buffer several times. No one does this anymore. I don't
feel I have to add extra steps by washing my tubes with distilled H20, and
we all know how expensive that stuff is these days ;-) If you are of the
paranoid variety, you can buy screw top tubes already silanized.

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
* - - - - - Methods FAQ list -> http://www-lmmb.ncifcrf.gov/~pnh/ - - - - - - *
* - - -  Anonymous FTP from ftp.ncifcrf.gov as file pub/methods/FAQlist - - - *
*******************************************************************************



More information about the Methods mailing list