6 Denaturing Gradient Gel Electrophoresis
93
Note: Short runs at higher voltage produce sharper bands. Therefore, we
prefer runs at 250 V for 2.5 hours.
ISA. Disassemble the apparatus and stain the gel (see "Staining").
Casting a parallel DGG
SB. See step 5A.
6B. Assemble the glass plates and 0.75 mm spacers in the gelcastingstand
according to the manufacturer's instructions.
7B. Prepare the two gradient solutions. The gradient range depends on the
melt map of the fragment to be analyzed (see "Optimizing Conditions"). Any denaturant concentration can be prepared by mixing
the Oo/o and 80% stock solutions. For a 16 x 18 x 0.075 cm gel, 12
ml of each concentration should be prepared in 15 ml tubes (e. g. Falcon). Various concentrations can be prepared according to the following table:
Desired denaturant concentration (%)
10
15
20
25
30
35
40
45
50
55
60
70
0% Stock 10.5 9.7 9.0
8.2 7.5 6.7 6.0 5.3 4.5 3.7 3.0
1.5
80% Stock 1.5 2.3
3.0 3.8 4.5 5.3 6.0 6.8 7.5 8.3 9.0
10.5
SB. To each tube, add 70 J..1l10o/o ammonium persulfate, mix gently, and
add 7 J..ll TEMED. Mix gently.
9B. Carefully pour the high er concentration solution into the chamber of
the gradient maker which is closest to the exit. Open the stopcock connecting the two chambers so that the solution fills the passageway,
then close again. Add the lower concentration solution to the second
chamber. Place the gradient maker on a magnetic stirrer elevated 25
cm above the bench. Stir the chamber next to the exit at medium velocity. Mixing should not introduce bubbles into the solution. Position the gel casting apparatus upright and insert the 20 gauge needle in
between the two glass plates. Carefully open the stopcock between the
two chambers, and the stopcock at the exit of the gradient maker. Adjust the flow so that the gel is poured within 4 minutes.
93
Note: Short runs at higher voltage produce sharper bands. Therefore, we
prefer runs at 250 V for 2.5 hours.
ISA. Disassemble the apparatus and stain the gel (see "Staining").
Casting a parallel DGG
SB. See step 5A.
6B. Assemble the glass plates and 0.75 mm spacers in the gelcastingstand
according to the manufacturer's instructions.
7B. Prepare the two gradient solutions. The gradient range depends on the
melt map of the fragment to be analyzed (see "Optimizing Conditions"). Any denaturant concentration can be prepared by mixing
the Oo/o and 80% stock solutions. For a 16 x 18 x 0.075 cm gel, 12
ml of each concentration should be prepared in 15 ml tubes (e. g. Falcon). Various concentrations can be prepared according to the following table:
Desired denaturant concentration (%)
10
15
20
25
30
35
40
45
50
55
60
70
0% Stock 10.5 9.7 9.0
8.2 7.5 6.7 6.0 5.3 4.5 3.7 3.0
1.5
80% Stock 1.5 2.3
3.0 3.8 4.5 5.3 6.0 6.8 7.5 8.3 9.0
10.5
SB. To each tube, add 70 J..1l10o/o ammonium persulfate, mix gently, and
add 7 J..ll TEMED. Mix gently.
9B. Carefully pour the high er concentration solution into the chamber of
the gradient maker which is closest to the exit. Open the stopcock connecting the two chambers so that the solution fills the passageway,
then close again. Add the lower concentration solution to the second
chamber. Place the gradient maker on a magnetic stirrer elevated 25
cm above the bench. Stir the chamber next to the exit at medium velocity. Mixing should not introduce bubbles into the solution. Position the gel casting apparatus upright and insert the 20 gauge needle in
between the two glass plates. Carefully open the stopcock between the
two chambers, and the stopcock at the exit of the gradient maker. Adjust the flow so that the gel is poured within 4 minutes.