Desalting reactions using Celite resin
Use of Celite resin to desalt Staphylococcus aureus reactions
1) Preparation of resin
Add 20ml of 3M potassium acetate pH4.8 to 67g of guanidine hydrochloride (GuHCl) and then add MilliQ water to give 90ml final volume. Add NaOH (10M) until pH is 5.5 then heat and stir to dissolve the GuHCl while maintaining the pH at pH5.5. Raise volume to 100ml. Filter the hot GuHCl solution through filter paper (some GuHCl may crystallise out during filtration/cooling) and add 10g Celite resin. The final GuHCl solution is approx. 7M.
Wash Buffer: 20 mM Tris-HCl, pH7.5, 200 mM NaCl, 5 mM EDTA diluted 1:1 with absolute ethanol.
STEB: Standard sample loading buffer: 40% (w/v) sucrose, 100 mM Tris-HCl, pH 8, 1 mM EDTA 0.5 mg/ml Bromophenol Blue
2) Desalting of reactions
Perform relaxation, decatenation or supercoiling reactions as normal (see appropriate datasheet). Once incubation of the reaction is complete, add an equal volume of well mixed Celite resin at room temperature. Vortex briefly to mix, leave 30 seconds, mix briefly and centrifuge 15 seconds, 13,000 rpm in a microfuge. Remove supernatant (discard) and wash pellet in 100ul Wash Buffer with mixing (a few seconds). Centrifuge 15 seconds, 13,000 rpm and remove supernatant (discard). Resuspend Celite in 1x STEB (usually 30ml), mix briefly, leave 1 min, mix briefly and spin 15 seconds, 13,000 rpm. Load supernatant on gel as normal, avoiding the Celite pellet.