magnetic bead losses during manipulation

W.Coco ifai at po.uni-stuttgart.de
Fri Jan 19 10:30:04 EST 1996


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?

 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. 

>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? 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?

Thanks again.

Wayne



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