C.B.S. Scientific DTSK-2401-220 - Manual (Page 48)

48
www.cbsscientific.com
3.13 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.
1. The following is a typical protocol for casting a 40%-60% denaturing gradient gel. See section 4.2 for stock
solutions. Refer to Section 3.5 Vertical Gradient Gel Casting 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 BACKFLOW 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”.
9. After polymerization rinse gel plate assembly with D.I. water to remove excess acrylamide or denaturants from
plate exterior.