Gel filtration spin columns

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[править] Home-made columns

[править] Preliminary

  • prepare collection tubes: cross-cut the cup of 1.7ml tube by scalpel (see figure)
  • prepare "filtered tips":
» cut by scalpel ~2.5x2.5mm2 squares of GF-C
» put GF-C square into a pipett tip and accurately push it to the end by a glass capillary
  • prepare suspension of "G-50, medium" in a buffer[1]
» prepare suspension: ~90% granules and 10% free liquid
» autoclave suspension in ~5ml aliquotes
» store at +4°C

[править] Preparation of columns

  1. mix well G-50 suspension (without bubbles!);
  2. put ~100µl of suspension into "filtered tip" and immediately shake it down[2]
  3. load suspension up to
    tipsuspension volume
    1ml (blue tip)1ml
    200µl (yellow tip)300µl
  4. now column is ready for use;
  5. optional, for storage:
    » close lower and upper ends with parafilm (in written order)
    » store at +4°C[3]

[править] Commercial columns

Illustra ProbeQuant G-50 Micro Columns; GE Healthcare Cat#28-9034-08

» these columns are in STE (100mM NaCl, 10mM Tris·Cl, 1mM EDTA) buffer

[править] Gel filtration

  1. (for home-made spin columns) remove parafilm before use in the following order: top, then bottom[4];
  2. put column into empty collection tube;
  3. spin: 0.5min at 2krpm;
  4. insert column into a new signed collection tube;
  5. load sample[5] and spin: 1min at 2krpm;

[править] Notes

  • do not exceed ~2.5krpm during centrifugation.

  • sample comes out of from the column in the "column preparation buffer". Either prepare columns in the appropriate buffer or (e.g. for commercial columns) change the buffer by washing the column by 3x volumes of the "new" buffer (under gravity).

  • NB! be careful if sample solution contains detergents: low molecular weight components may pass through the column in micelles.

  • it is possible to use different gel-filtration media. "G-50 coarse" and "G-50 fine" are also acceptable (but "medium" is more convenient).

  1. typical buffers:
    » STE - for removal of not incorporated labelled nucleotides
    » 1mM TrisCl, pH 7.0 — for purification of First-strand synthesis reaction
  2. to prevent formation of an air bubble in the end of the tip
  3. for RNA-applications it is better to prepare columns just before use; for DNA — it is possible to prepare a lot (~100) of columns at once and store them at +4°C for more than a year
  4. otherwise GF-C filter may be sucked into the column
  5. sample volume should be less than
    columnmax. sample volume
    1ml (blue tip)100µl
    200µl (yellow tip)30µl
    ProbeQuant G-5050µl

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