Bio-Rad ReadyPrep™ 2-D Starter Kit - Manual (Page 15)

a Remove the kit from the refrigerator and unpack the 2 bottles of equilibration buffer I
(WHITE or SILVER caps), the 2 bottles of equilibration buffer II (RED caps), the bottle
of 30% glycerol (CLEAR cap), the 2 bottles of iodoacetamide (RED caps), and the
bottle of overlay agarose (CLEAR cap).
b Remove the crimp and open the two bottles of equilibration buffer I and equilibration
buffer II. (NOTE: if 3 or fewer 17 cm IPGs, 5 or fewer 11 cm IPGs, or 8 or fewer 7 cm
IPGs are to be separated in the second dimension, then only 1 bottle each of equili-
bration buffer I and II and 1 bottle of iodoacetamide is needed.)
Prepare Equilibration Buffer I: To each one bottle of equilibration buffer I carefully
add 13.35 ml of the supplied 30% glycerol solution. Each bottle contains a stirbar. Place
the bottle onto a stirplate and mix until all the solids have completely dissolved. It may be
necessary to periodically swirl the contents of the bottle to dislodge solids remaining on
the walls of the glass bottle. Generally, the solids will be dissolved in less than 5 minutes.
The bottle will chill as the urea in the solids dissolves. To expedite this buffer reconstitution
process the bottle can be warmed slightly in the palm of the hand or placed into a water
bath set for 25-30ºC as the solution stirs. Do not heat above 30ºC.
Prepare Equilibration Buffer II: To each bottle of equilibration buffer II carefully add
13.35 ml of the supplied 30% glycerol solution. Each bottle contains a stirbar. Place the bottle
onto a stirplate and mix until all the solids have completely dissolved as described above for
equilibration buffer I.
4.7 IPG Equilibrations.
1. If the IPGs were frozen at -70ºC then confirm that the strips have thawed before continu-
ing. Frozen IPG strips containing sample are opaque white and turn to clear after thawing
and redissolving of the urea present inside each strip.
2. Using Table 3 as a guide, add the indicated volume of equilibration buffer I to each chan-
nel containing an IPG strip (gel side up per section 4.4).
3. Place the tray on an orbital shaker and gently shake for 10 minutes. Select a slow shaker
speed to prevent the buffer from sloshing out of the tray.
4. After placing the IPG strips to shake in equilibration buffer I, proceed to complete the
preparation of equilibration buffer II. Add the contents of one bottle of iodoacetamide to
each bottle of equilibration buffer II. Return the bottle(s) to stir until the iodoacetamide is
fully dissolved.
5. At the end of the 10 minute incubation, discard the used equilibration buffer I by carefully
decanting the liquid from the tray. Decanting is best carried out by pouring the liquid from
the square side of the rehydration/equilibration tray (left side in Figure 4), until the tray is
positioned vertically. Take care not to pour out the liquid too quickly at first as the strips
may slide out of the tray. When most of the liquid has been decanted, “flick” the tray a
couple times to remove the last few drops of equilibration buffer I.
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