spun column

[David Micklem]

In article <33E8A0DA.3ADF71DF at unity.ncsu.edu>, Tom Phelan
<tjphelan at unity.ncsu.edu> wrote:


>
>I use this protocol frequently, and it works well.  Don't ask where it
>originated, I sure can't answer that.

To give some idea of the possible variations of this protocol (see also the
FAQ under removing dye-terminators...)
>
>1. Poke a hole in the bottom of a 0.5 ml microfuge tube using a 16 gauge
>needle.  Plug the hole with a small amount of silanized glass wool
>(cotton soaked in 70% ethanol also works well).

Or, nylon aquarium filter material (like cotton-wool), or small glass beads
(eg No.11 glass beads from Jencons, around 200um), or even (shock!) a bit
of Kimwipe paper tissue.  I use the glass beads, and never found it
necessary to siliconize them. (But my application is slightly different...)



>2. Load the 0.5 ml tube inside a 1.5 ml tube.  Fill the 0.5 ml tube with
>700 microliters of Sephadex G-50 (swelled in TE and autoclaved).

>3. Spin 2 minutes at 1000g.  Add 50 microliters of TE and re-spin.
>Repeat until only 50 microliters of TE is recovered in the 1.5 ml tube.
>(this usually takes 2 or 3 spins).

Make sure that you discard the flow-through each time, or the bottom of
your mini-column will end up immersed, and it'll NEVER come right!  I find
that if you  use a screw-cap 1.5ml tube, the 0.5 is held higher up, and a
single 50ul spin through is sufficient. YMMV

>4.  Replace the 1.5 ml tube with a clean 1.5 ml tube.  Load your
>labeling reaction and spin at 1000g for 2 minutes.  (Your labeled probe
>is in the eluate, the unincorporated nucleotides are retained on the


Don't forget to monitor the centrifuge afterwards!  My guess is that this
method, with open-topped tubes, _could_ end up spraying a fair bit around
(but I've not used one of these columns for hot samples). I'd also leave
the column standing for a minute or so after adding the sample to let it
soak in....


David

-- 
D.R.Micklem,                                Time flies like an arrow... 
Wellcome/CRC Institute,            Fruit flies like a banana.       
Cambridge CB2 1QR, UK               Email:drm21 at mole.bio.cam.ac.uk
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