Bio-Rad ReadyPrep Sequential Extraction Kit - Manual (Page 6)

necessary to hold the vial in a bath of warm (tap) water for
more efficient warming. Rehydrated Reagent 3 consists of 10
ml of 5 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (w/v) SB
3-10, 40 mM Tris, and 0.2% (w/v) Bio-Lyte 3/10 per vial.
Storage of rehydrated solution 1, 2, 3. Following
rehydration, aliquot the solutions as convenient and store the
aliquots at -20 °C or lower. A convenient aliquot size is 1 ml,
each placed into a microcentrifuge tube for storage. Thaw the
frozen aliquots prior to use and discard unused portions of
each aliquot. It may be necessary to warm the solution of
Reagent 3 to disperse the SB 3-10 detergent.
Reducing Agent TBP. Tributyl phosphine (TBP) has an
unpleasant odor and is very volatile. Work with it in a fume
hood. Wear a laboratory coat and gloves when handling the
ampoule of tributyl phosphine. Change gloves often. Wipe up
spills with wet towels. Open the ampoule by snapping the top
off at the scored neck. Transfer the entire contents of the
ampoule to the screw-top vial provided. Screw the cap of the
vial down tightly and store the vial at -20 °C or lower to
prevent evaporation of the TBP.
Extraction Solutions. Before carrying out an extraction,
mix 10 µl of TBP solution with every 1 ml of Reagent 2 and
(or) Reagent 3 to be used in the extraction (1:100 dilution).
TBP is not used with Reagent 1. Use only polypropylene or
glass pipettes when distributing the reducing agent TBP
concentrate. Most standard pipette tips, such as those sold
by Bio-Rad, are made of polypropylene. Do not use
polystyrene pipettes, since they will dissolve in the NMP solvent.
4
4006167b.qxd 08/28/2000 6:43 PM Page 4