wmelchior at ntet.nctr.fda.gov
Mon Jun 28 10:22:02 EST 1993
In article <1993Jun25.230351.1370 at Princeton.EDU>,
lpcasson at phoenix.Princeton.EDU (Lawrence P. Casson) writes:
> I'm looking for an inexpensive way to purify the products of a
> cycle-sequencing reaction from dye-terminators (from ABI sequencing
> kit). As I need to sequence a hundred or so mutants of the same
> sequence, I wonder if the purification can be done for much less than
> $3.00 per reaction (the cost of spin columns from Princeton Separations
> which work quite well for me - I don't know what's in them).
There have been a couple of postings on homemade spin columns:
1) From: bsh at med.pitt.edu (Basavaraju Shankarappa); 19 Nov 92
>We now routinely purify the excess dye from reactions using home (lab)-made
>quick spin columns. They may not overcome the tedium but they sure are
>cheap. We make a hole in the bottom of 0.5 ml eppendorf using a 26 g
>needle and add about 25 ul volume of zirconium glass beads. Then I pour
>sephadex G-50 in 0.3M NaAc (5g in 60 ml) to the top of the tube. Stick the
>small eppendorf inside a 2 ml tube and spin at about 500 rpm for couple of
>minutes. Now the tube is transferred to a new 1.5 ml tube and the sample is
>added on top. Now I spin at about 1500 rpm for couple of minutes. The
>eluate is precipitated by atleast 3 volumes of isopropanol washed with 70%
>etoh and resuspended for loading.
>bsh at med.pitt.edu
>Univ of Pittsburgh
[This is the method given in Paul Hengen's Methods FAQ. The FAQ also has a
brief discussion, with reference, of using static columns.]
2) My approximate retyping of a post from Michael Benedik,
bchs1b at ELROY.UH.EDU; 20 Nov 91:
>I make homemade spin columns in a 1 ml syringe with a little glass wool
>stuffed in the bottom (can use silicon wool if you want to be sure it is
>not sticky) and fill the syringe full of column matrix. Can use Sephadex
>G-50; we prefer BioRad P-10. Put the column in a 15 ml Falcon tube, spin
>at 2,000 rpm for 2 minutes; the matrix should now look dry. Cut the lid
>off a 1.5 ml microtube and put in the bottom of Falcon tube, put syringe
>end inside 1.5 ml tube, load sample, and spin at 2,000 rpm for two minutes.
>I have found this really only works reliably in a swinging bucket rotor.
>Speed and time are variable, but do both spins the same. Discard syringe
>and your sample is in the microtube. This matrix works well for desalting
>and removing nucleotides. If you want to remove linkers, you can use other
>matrices; some commercial spin columns use CL2b, CL4B and CL6B to separate
>larger oligos from each other. I have no experience with those, but they
>should work. Just use a strong bead like an acrylamide bead so that it
>does no crush like agarose beads do.
3) A couple of additional notes: I've not had much experience yet, but
have succesfully used columns based on Shankarappa's method. I used about
25 microliters of pure sand in the bottom rather than glass or zirconium
beads. Spinning at full speed in a microfuge did not work well (My
microfuge does not have adjustable speed.), so I've used a swinging bucket
centrifuge at lower speed, as suggested by Benedik. Aside from the time
involved, which isn't really very much, these columns are MUCH cheaper than
the commercial ones.
If Michael Benedik is reading, perhaps he can comment on why he prefers
P-10 to G-50.
Views expressed are not necessarily those of NCTR, its sponsoring agencies,
or the United States government.
Bill Melchior ||
National Center for Toxicological Research || ALL statements
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|| are false.
WMELCHIOR at NTET.NCTR.FDA.GOV ||
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