spun column

Tom Phelan tjphelan at unity.ncsu.edu
Wed Aug 6 11:05:47 EST 1997


Molecular Pathology Lab wrote:

> Hello all-
>         I was wondering in anyone has a protocol for doing a G-50 spun
> column
> using a microcentrifuge for seperating unicorporated from labled
> probe.
> I have used the 1mL (tuberculin) syringe method, but I don't have
> access
> to a clinical centrifuge, but I do have access to an old
> microcentrifuge.  Any help that you can give me will be greatly
> appreciated, and you can either send it to my e-mail or to the news
> group.
>
> Thanks in advance.
>
> -Eric W. Olle

  Hi Eric

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

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).
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).
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
column)
(Note:  It's usually a good idea to make one or two more columns than
you need, in case one of the columns gets messed up.)

I hope this is helpful.  If I have left out any information, or you have
any questions, please feel free to email me.

Tom Phelan




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