magnetic bead losses during manipulation

Paul N Hengen pnh at cockleberry.ncifcrf.gov
Fri Jan 19 12:56:56 EST 1996


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

: Dear Paul,
: Thanks for all the tips. May I ask for (and offer one) clarification?

| 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.

: This seems cumbersome, do you have an efficient method for doing lots at 
: once? How exactly do you silanize? You mention "until" the NaOH step
: is silane incompatible with base?

It's really not that bad. You use one large tip or disposable pasteur pipette
and squirt in about 1ml of sigmacote, suck it back in and move to the next tube
in the rack....couldn't be easier. For tips, have one tube of sigmacote open
and suck in the maximum volume, dispense back into the tube, and eject the tip
into a new rack. I dry everything under the hood.

I wouldn't use silanized tubes when doing the NaOH reaction because this
is how you remove the silane coating -> the silane could interfere with
sequencing, enzyme reactions, etc.

: 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?

: I didn't mean to imply that they recommend against BSA, only that they
: include it in the first washes and not thereafter (I just checked the
: latest manual and they don't include it in at least some initial washes). 
: In any case, I am sure BSA doesn't bother. 

Well, I have experience with BSA sticking to the streptavidin which can
then come off when boiled. We've proved this by isolating BSA using only
beads, extensive washing, boiling, running SDS-PAGE. I was thinking you
or Dynal knew about this already and had an explanation. I now do all wash
steps without BSA. I don't see any reason to include it.

| 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.

: Hmm. Only the non-biotinylated strand gets stripped, right?

Yes.

: I thought the 
: biotinylated strand stays on unless you get really tough and I think one
: can still use the boiled beads with their ssDNA as a capture strand and 
: magnetically separate as usual, etc. Is this wrong?

You are right. That's what I meant.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
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