Instruction manual

6.4 Casting Parallel DGGE Gels

1. Position the gel assembly by standing it upright (Figure 6.8).

Note: Place a comb into the sandwich before casting the gel. Placing a comb after cast-

ing the gel will disturb the gradient.

Fig. 6.8. Pouring a parallel gradient gel.

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.

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

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

4. Repeat steps 4 through 8 in Section 6.3.

5. Place the high density syringe into the syringe holder (high density side for top filling).

Make sure to place the syringe in the correct syringe holder, i.e., the placement of the

low and high density syringes depends on whether the gel is being poured from the top

or bottom. Note: Parallel gradient gels are top filled. Check for proper high and low

syringe position on the gradient delivery system. Hold the syringe by the plunger and

insert the lever attachment screw into the lever groove. Try not to handle the syringe, it

may dispense the gel solution out of the syringe. Carefully position the lever attachment

screw by placing it in the groove and sliding the syringe back towards the cam side.

Tighten the syringe holder screw against the syirnge sleeve. Do the same for the low den-

sity solution (low density side for top filling).