Manual

21
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3.3 Vertical Gradient Formation
To determine the range of gradient appropriate for your fragment analysis,
please read the enclosed paper by Myers, Shefeld 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% gradient gel. Refer to
Section 3.2 Vertical Gradient Gel Casting, gure 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 (C-2),(GM-40) as
shown in g. 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 rst (V-1), then outside (V-2) valve to start gravity
ow. 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 ll sandwich, use 0% to "top-off". If using water saturated butanol
for overlay, leave 0.5cm void to create at interface.
9. Insert the sample well comb. Allow the gel to polymerize for 20-30
minutes.
Go to Section 4 for Gel Running Preparation.