Acrylamide Prep
    per Adam (8/2004)



    The acrylamide gels described in this prep are used to separate fragments of DNA that differ in size even by two nucleotides. The final concentration of these gels is 14%, but this depends on the amount of time and voltage a gel is run and overall length of the fragments. The typical time and voltage is 280V for 140 minutes.

    1. Prepare the acrylamide gel apparatus for pouring.


    (a) Assure that no dry acrylamide remains in main compartment.
    (b) Place orange rubber pad in bottom of main compartment.
    (c) Place a piece of wax paper on the back of the main compartment and add a thick plastic plate.
    (d) Place a piece of wax paper on the top of the plastic plate.
    (e) Add a glass plate with a flat-edged top.
    (f) Next, add 2 gray spacers: one on each side of plate.
    (g) Place a plate with a concave-edge on top of the spacers.
    (h) Add 2 more gray spacers on top of previous plate; same as before.
    (i) Place a plate with a flat-edge on top of the spacers and place a piece of wax paper on top of it.
    (j) Repeat steps e through i until the main compartment is nearly full. At this point there should be 14 gaps for individual gels or a total of 7 gel 'sandwiches'.
    (k) Additional, thin plastic plates are used to fill the remaining space. Typically, 3 or 4 will suffice; no wax paper is needed between these.
    (l) The face of the gel apparatus should be added next. Once it is in place, assure the foam seal is still intact. Also, DO NOT tighten the plastic screws.
    (m) Next, locate 4 red plastic clips and clamp the face to the main compartment. The screws can tightened now.
    (n) Use the 'SpaceMate' to make certain the spacers are evenly spaced.
    (o) Add combs to the apparatus. Ensure that each gap has a comb in it.
    (p) Place the assmebled apparatus next to the gel loader and attach the tubing from the loader to the tube in the front of the apparatus.

    2. Prepare the acrylamide solution in a 500 mL beaker. Add ingredients according to the recipe below:

     
    Acrylamide Solution 
    ddH20
    40% Acrylamide
    50X TAE
    10% APS
    TEMED*
     
    189 mL
    84 mL
    5.6 mL
    2.1 mL
    168 microliters

     

    *Add TEMED last. It catalyzes the polymerization of acrylamide to polyacrylamide.

    3. Mix ingredients by swirling. Be careful not to create bubbles.

    4. Bottom-load the apparatus using the Easy-Load maching. DO NOT increase the loading speed past the mark on the dial; doing so will cause the tubing to become entangled inside the machine. This will prevent smooth flow and the acrylamide will polymerize and harden before the apparatus is filled completely rendering all work done up to this point useless. IMPORTANT: While the acrylamide is being loaded, ensure the tubing remains submerged in the acrylamide solution. If it is taken out, air will enter the apparatus and form bubbles in the gels.

    5. Once the entire acrylamide solution has been loaded, fold over the tube on the face of the gel: this will prevent backflow. Remove the tubing from the Easy-Load machine. Continue to hold the tube from the apparatus in a pinched state. Now, take a metal binder clip and reinforce the pinched tube.

    6. Clean out the Easy-Load machine by running water through the tubing.

    7. The gels will need to sit for ~1.5 hours for the acrylamide to completely polymerize.

    8. Disassemble the apparatus and remove and clean the gels.

    (a) Loosen the screws on the face of the apparatus.
    (b) Remove the red clips.
    (c) Remove the face and thin plastic plates.
    (d) Removing the gels can be tricky; slide a razor blade between the first gel 'sandwich' and the second. Press the razor backwards. The gel 'sandwich' will separate from the others. Once there is a small separation, you cn use your fingers to pry the 'sandwich' out.
    (e) Now pull the combs straight out, slowly.
    (f) Once the gel 'sandwich' has been removed, it needs to be cleaned. Use the razor to scrape excess acrylamide at the top of the gels is removed with the razor by sliding it in and pressing back on it; the excess gel can be lifted up with the razor and removed. All acrylamide should be discarded in the trash.
    (g) Wash the 'sandwich' in tap water. This removes any acrylamide that was unable to be removed by the razor.
    (h) Set the cleaned 'sandwich' aside for the time.
    (i) Repeat d through h until all 'sandwiches' have been removed and cleaned.
    (j) Use the razor to remove the plastic plate. Clean the main compartment with the razor similar to the gels. Was all parts of the gel apparatus with tap water and place in drying rack. The tube on the face plate can be cleaned with a syringe needle, and flushed out with a syringe.

    9. Lay a large sheet of PVC (saran wrap) on a flat surface. Place a paper towel in the middle of it. Pour some 1X TAE solution on the paper towel. Now place a single gel 'sandwich' on the paper towel. Fold the edges of the towel so that they cover the sides of the gel. Wrap the 'sandwich' in the PVS and store at 4 degrees Celsius.