Instruction manual

6.2 Model 475 Gradient Delivery System

The Model 475 Gradient Delivery System is used with the D GENE system to construct

reproducible linear polyacrylamide gradient gels. Refer to the Model 475 Gradient Delivery

System instructions for information on its set-up and use. The Model 475 Gradient Delivery

System has a 7–50 ml capacity, making it ideal for the construction of 16 x 16 cm or 7.5 x 10 cm

polyacrylamide gradient gels. The mixing of the high and low density solutons occurs in the

Y-fitting and a single outlet leads to the gel sandwich. The gradient may be cast from the top

for parallel gels or from the side for perpendicular gels.

6.3 Casting Perpendicular DGGE Gels

1. Position the gel assembly tilted to about a 20° angle by adjusting the rod on the comb

gasket. This is to insure that any air bubbles that form while casting the gel escapes to the

air vent (Figure 6.7).

Fig. 6.7. Pouring a perpendicular gradient gel using the Model 475 Gradient Delivery System.

2. Refer to the Model 475 Gradient Delivery System manual for details on set-up and

operation of the gradient former.

3. Insert and fasten all three metal fittings into the Y-fitting. Connect the free end of the

9 cm Tygon tubing to one of the metal fittings in the Y-fitting and connect to the other end

a luer coupling. Connect each of the luer fittings on the two long pieces of tubing, then

connect the luer fitted ends to same size syringes. Do not connect the long Tygon tubings

to the Y-fitting.

4. Label one of the syringes LO (for the low density solution) and one HI (for the high den-

sity solution). Attach a plunger cap onto each syringe plunger “head.” Tighten enough to

hold the plunger in place. Slide each syringe into a syringe sleeve. Twist and position the

sleeve to the middle of the syringe with the volume gradations visible. Make sure that

the lever attachment screw is in the same plane as the flat or back side of the sleeve. This

is extremely important for proper attachment of the syringe to the lever.

Note: Ensure that the tubing pieces are free of any gel material by simply pushing water through

the tubing with the syringe. The tubings should be free of any materials before casting.

5. Rotate the cam wheel counterclockwise to the start position. At the start point, the lever is in

the vertical position. Adjust the volume adjustment screw to the desired setting. Refer to the

Model 475 Gradient Delivery System instructions, Section 4.1, for the correct setting to use.