C.B.S. Scientific DGGEK-2401 - Manual (Page 23)

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3 5 Perpendicular Gradient Formation
To determine the range of perpendicular gradient appropriate for your fragment
analysis, please read the enclosed paper by Myers, Sheffield and Cox, as well
as the Methods and Enzymology V. 212 paper by Abrams and Stanton. This
gives you an excellent overview of the determination of melting behavior of your
fragments.
2. The following is a typical protocol for casting a 40%-60% gradient gel. Refer to
Section 3.2 Vertical Gradient Gel Casting, figure 3.5 for apparatus assembly. In
an ice bucket, place two 50ml conical tubes labeled “A” and “B”. Add to tube “A”
• 11.5ml of 40%/7.5%
• 80 µI (10%) APS
• 5 µl TEMED
2. Add to tube “B”:
• 11.5ml of 60%/7.5%
• 80 µI (10%) APS
• 5 µI TEMED
3. Pour solution "B" into right side of gradient maker,(GM-40), and open interior
valve to allow air bubble to escape. Let as much as 1 ml "B" solution BACK-
FLOW into left side of gradient maker. Decant the 1ml back into right side with
pasteur pipette. Remove any residual solution with absorbent paper.
4. Add solution “A” to left side of gradient maker.
5. Turn on magnetic stirrer.
6. Exit tube should be attached to near side of gel plate with tape.
7 Open inside (V-1) valve first, then outside (V-2) valve to start flow.
8. Gel volume is 23ml, using the 0.75mm spacers. Allow 20-30 minutes for gel
polymeraztion. If gel volume is not enough to fill gel sandwich, use 0% to “top-
off”.
Go to Section 4 for Gel Running Preparation