Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 30)

7. Remove the alignment card. Remove the sandwich assembly from the casting stand and
check that the plates and spacers are flush at the bottom. If the spacers and glass plates
are not flush, realign the sandwich and spacers to obtain a good seal (Repeat steps 5–7).
8. When a good alignment and seal are obtained, tighten the clamp screws until it is finger-tight.
Casting Parallel Denaturing Gradient Gels
1. Place the gray sponge onto the front casting slot. The camshafts on the casting stand should
have the handles pointing up and pulled out. Place the sandwich assembly on the sponge
with the shorter plate facing you. When the sandwich is placed correctly, press down on
the sandwich and turn the handles of the camshaft down so that the cams lock the sandwich
in place. Position the gel sandwich assembly by standing it upright.
2. One length of Tygon tubing is provided and should be cut into two 15.5 cm lengths and one
9 cm length. The longer pieces of Tygon tubing will be used to conduct the gel
solution from the syringes into the Y-fitting. The short piece of Tygon tubing will conduct the
gel solution from the Y-fitting to the gel sandwich. Connect one end of the 9 cm Tygon
tubing to the Y-fitting and connect a luer coupling to the other end of the 9 cm tubing. Connect
luer fittings onto the two long pieces of tubing. Connect the luer fittings to 30 ml syringes. Do
not connect the long Tygon tubing to the Y-fitting at this time.
3. Label one of the syringes LO (for the low density solution) and one HI (for the high
density solution). Attach a plunger cap onto each syringe plunger “head.” Position the
plunger "head" in the middle of the plunger cap and tighten enough to hold the plunger
in place. Position the cap in the middle for proper alignment with the lever on the
gradient delivery system. Slide each syringe into a syringe sleeve. Move the sleeve to
the middle of the syringe, keeping 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 very
important for proper attachment of the syringe to the lever.
Note: Insure that the tubing is free of any gel material by pushing water through the
tubing with the syringe. The tubing should be free of material before casting, remove any
remaining water from the tubing.
4. Rotate the cam wheel counterclockwise to the vertical or start position. To set the desired
delivery volume, loosen the volume adjustment screw. Place the volume setting indicator
located on the syringe holder to the desired volume setting. Tighten the volume adjustment
screw. For 16 x 16 cm gels (1 mm thick), set the volume setting indicator to 14.5. Refer to
Section 4.1.
5. From the stocks solutions, pipet out the desired amounts of the high and low density gel
solutions into two disposable test tubes (refer to the Section 4.1).
Optional: To visually check the formation of the gradient, add 100 µl of DCode dye
solution per 5 ml high density solution.
The steps below are time-sensitive (about 7–10 minutes). Insure that steps 2
through 5 are done before proceeding further. Be thoroughly familiar with the
following steps before casting the gel.
6. Add the final concentration of 0.09% (v/v) each of ammonium persulfate and TEMED
solutions. The 0.09% (v/v) concentrations allow about 5–7 minutes to finish casting the gel
before polymerization. Cap and mix by inverting several times. With the syringe connected
to the tubing, withdraw all of the high density solution into the HI syringe. Do the same for
the low density solution into the LO syringe.
Note: Acrylamide is a very hazardous substance. Use caution: wear gloves and eye
protection at all times. Avoid skin contact.
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