magnetic bead losses during manipulation

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Wed Jan 17 12:56:23 EST 1996


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

: 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. 

You must start with more than 5 ul in my experience. I use 15-20 ul to be sure
I can see the pellet.

: 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 have settled on at least 3 minutes! Some of the particles are invisible.

: 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 something I should not (i.e. technique issues).

I have had the same problem. I solved it by treating all my plastic tips and
tubes with sigmacote (silanizing agent) before use. Maybe you should use
siliconized tubes until the last step (NaOH). I also use screw-top tubes
to avoid the eppi-"pop" which jars the contents, swirling it around the pellet.
I pipette out liquid while the tube stands in the magnetic tube holder.

: 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. 

Why do they not recommend BSA???? Do you know?

: 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?

I assume static attraction can be to blame.

: 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?

I've stored attached DNA-beads for more than a year in TE at 4C. The loss of
DNA mostly occurs when multiple pipettings are performed, which is presumably
due to abrasion. Dynal beads are better than Promega because they are round
and Promega's are blobules (very irregular). Therefore Dynal beads+DNA last
longer through many manipulations, but are more expensive. I pay the extra
because prep time is more important to me.

: 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)?

We boil them to strip ssDNA off. The beads are fine, but the DNA is obviously
now ss on them. Of course the streptavidin is no good afterward.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
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