Bio-Rad Model 491 Prep Cell and Mini Prep Cell - Manual (Page 14)

9
3. It is advisable to cool the gel during polymerization. Cooling prevents ex cess heat accumulation in the
interior of the reaction mixture and aids in the formation of uniform gels. To cool, pump room temperature
water (or buffer) from an external source through the cooling core. Ensure that cooling is in progress prior
to casting the gel.
Fig. 7. Diagram of cooling path during polymerization. Cooling of gels during poly merization is recommended. This is accomplished
by circulating room temperature buffer between a reservoir and the cooling finger using the buffer recirculation pump.
4. Prepare the acrylamide monomer solution. Refer to Section 4 for selecting the appropriate acrylamide
gel concentration for a given application. Sections 8 and 9 describe preparative gel for mulations for
SDS-PAGE and native-PAGE, repsectively.
5. Pour the degassed monomer mixture into the gel tube through the gap be tween the cooling core and
the collar of the gel tube assembly. Avoid trap ping air bubbles in the gel. Gently tapping the casting
stand (with the gel tube assembly mounted to it) against the bench top will help to dislodge trapped air
bubbles. Visually inspect the gel for bubbles immediately after pouring the separating-gel solution into
the tube.
Carefully overlay the resolving gel with water-saturated 2-butanol or tert-amyl alco hol using the narrow
polytetrafluoroethylene (PTFE) tube affixed to a syringe (provided with the unit). Allow the resolving gel to
stand overnight for complete polymeriza tion (catalyst con centration in resolv ing gel is 0.025%
APS/0.025% TEMED). After 1–2 hours polymer ization, re place the alcohol overlay with gel buffer. In the
case of SDS-PAGE and Ornstein-Davis nondenaturing gels, this should be 0.375 M Tris/Cl, pH 8.8
buffer.
6. Very carefully decant or aspirate the buffer overlay. Cast the stacking gel, approx imately twice the
sample volume, on top of the resolving gel. Overlay the stacking gel monomer with water-saturated
2-butanol or tert-amyl alco hol. Allow the stack ing gel to polymerize for 1–2 hours.
9