C.B.S. Scientific TTGEK-2401-220 - Manual (Page 42)

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3.7 Vertical Gradient Formation for DGGE.
To determine the range of gradient appropriate for your fragment analysis, please read the
enclosed paper by Myers, Sheffield and Cox, especially section 6.1.3 through 6.2.1 (pages 124
to 126). 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 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 (C-2),(GM-40) (Fig. 3-5), 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 1 ml back into right side with pasteur pipette. Remove any residual
solution from (C-1) with absorbent paper.
4. Add solution "A" to left side of gradient maker.
5. Turn on magnetic stirrer.
6. Exit tube with needle should be secured to gel plate notch with tape.
7. Open inside valve first (V-1), then outside (V-2) valve to start gravity flow. Optionally, Turn on
mini-pump and pump acrylamide at medium speed. Determine optimal speed empirically.
8. Gel volume is 23ml, using the 0.75mm spacers. If gel volume is not enough to fill sandwich,
use 0% to "top-off". If using water saturated butanol for overlay, leave 0.5cm void to create flat
interface.
9. Insert the 16-well rectangular tooth comb. Allow the gel to polymerize for 20-30 minutes.
10. If using Multi-Gel Casting Chamber please refer to instructions at bottom of page 33 under
Disassembly.