Bio-Rad DCode™ Universal Mutation Detection System - Manual (Page 26)

Casting Perpendicular Denaturing Gradient Gels
1. 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 10 ml or 30 ml syringes.
Do not connect the long Tygon tubing to the Y-fitting at this time.
2. 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.
3. 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 7.5 x 10 cm gels (1 mm thick), set the volume setting indicator to 4.5. For
16 x 16 cm gels (1 mm thick), set the volume setting indicator to 14.5. Refer to Section 4.1.
4. From the stocks solutions, pipet out the desired amounts of the high and low density gel
solutions into two disposable test tubes, 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.
Note: The gel solution volume should be greater than the amount set on the volume
adjustment lever. For example, if the setting indicator is set at 4.5, the syringe should
contain 5 ml of the gel solution. This extra solution is needed to push the correct amount
into the gel sandwich.
The steps below are time-sensitive (about 7–10 minutes). Insure that steps 1 through
4 are done before proceeding further. Be thoroughly familiar with the following steps
before casting the gel.
5. Add a 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.
6. Carefully remove air bubbles from the LO syringe by turning it upside down (plunger cap
towards the bench) and gently tapping. Push the gel solution to the end of the tubing. Do
not push it out of the tubing as loss of gel solution will disturb the volume required to cast
the desired gel.
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