magnetic bead losses during manipulation
ifai at po.uni-stuttgart.de
Wed Jan 10 07:36:06 EST 1996
I am working with Dynal's M-280 streptavidin coated magnetic beads and
am having a problem. The problem entails a significant loss of beads during
manipulation. I started with 5 ul (50 ug) of beads, enough to see a quite turbid
solution and large pellet when the magnet was applied. However, after the
following manipulations, there was only a small fraction of the beads
visible: I washed the beads 2x in 1 M NaCl and bound a 100 bp, gel
purified, dsDNA biotinylated PCR product at 500 mM NaCl. I then washed 3x
with H2O and eluted in 0.1 N NaOH 10 min, washed in same and finally
neutralized with three washings (one with 200 mM tris, pH 8, two with TE)
and resuspended in TE.
Each time I attract with the magnet, I leave the tube in the magnetic stand for at
least a minute and pipette away the supernatant carefully without touching
the magnet side of the tube. I also re-add buffer within 10-20 s, so as not
to let the beads dry out. Each washing is with at most 120 ul, and more
typically, 30-50 ul.
A small fraction of the beads appear to adhere somewhat to the sides of
the tube in just about every step after the NaOH treatment. That is, they
cannot be washed away even with vigorous pipetting, but can be scraped off
(some may end up back in solution, some may stick to the disposable pipette
tip). The losses are significant. My guesses are as follows. Perhaps I am doing some-
thing I should not (i.e.
technique issues). For example, I only washed the beads 2x with 1 M NaCl
before use (as recommended in CPG protocol sheets) instead of the PBS+BSA
wash followed by 2x with salt Dynal recommends. I assume this could not be the
source of the
problem, especially since I use the beads immediately and Dynal doesn't
recommend BSA in further steps. Another possibility is that our Eppendorf-
type microcentrifuge tubes (1.5 ml capacity) have some strange surface or
coating that adsorbs beads. The observed losses coincided with low salt
washings; can ionic strength play a role? Any other suggestions?
Two additional questions:
1. Are there losses in performance when beads are stored after DNA is bound?
Can I store them in TE? How long can they be stored?
2. Does anyone have data on the heat stability of M-280 (or other) beads that have
biotinylated DNA bound (i.e. loss of function at various temps vs. time,
effects of salt concentration)?
Sorry for the long wind.
Thank you for your help.
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