Cleaning Big Dye reaction with Sephadex G-50
Materials:
- Sephadex G-50 DNA grade (fine; available through many distributors including Pharmacia and Sigma.
G-50 Med or Fine also works)
- Columns (96-well or used AutoSeq G-50 columns from Amersham).
- Autoclave
- Centrifuge
Method:
- Determine the number of columns that will be used.
- Weigh out the sephadex and put it in an autoclave safe glass bottle.
(use 2 g. Sephadex G-50 with 80 ml. of water) - Autoclave the mixture for 15 minutes and store in a refrigerator.
Note: Autoclaving will speed up the hydration of the sephadex and kill any fungus that may show up if the slurry is not used immediately. - Decant excess water after autoclaving so that there is approx. 1/3 water with 2/3 sephadex.
- Transfer 750 ul of well shaken slurry to the top of each of the columns.
Note: Make sure the slurry is thoroughly mixed and uniform before adding it to a column. If this is not done, the sephadex "pillars" may not be uniform and the results from the sample clean-up may not be good. - Spin down the G-50 column at 2000xg (5000 rpm in Eppendorf microcentrifuge) for 1 minute to
remove the water (if using a standard microfuge with variable speeds a setting of 5 out of 14
works well). Spin the 96-well plates at 750xg for approximately 2 minutes.
Note: The columns should be spun in a tube that it will collect the water from the hydrated sephadex. A waste tray should also be used for the 96-well tray. - Transfer the columns to either the sample collection tray (96-well) or a 1.5-ml labeled
microfuge collection tubes.
- Add the sample to the middle of the sephadex bed.
- Spin the sample again at the same speed and time as in step 6.
- Dry down the sample.
- The remaining resin can be tapped out and the column re-used indefinitely after washing with tap water and rinsing with dH2O. For the 96 well columns, it may be necessary to spray out some of the chambers to remove all the used matrix.
Protocol adapted from
https://dna.biotech.wisc.edu/documents/Non-bead_cleanups.htm