Assembly of the BioRad Maxi-Vertical Slab
Gel Electrophoresis Apparatus

This is NOT about the mini-gel apparatus.

Make a practice "dry" run through all the following steps of assembly before doing it for real. The following steps contain a lot of words for a procedure that takes only a couple of minutes to accomplish.

Assembly of the glass panes
1. Take two glass panes. They should be clean and DRY. Each pane should have at least one LONG edge that is free of chips that extend the thickness of the glass. Call such a non-chipped edge "top."
2. Lay one pane on your countertop with the "top" away from you.
3. Pick up a 2 mm spacer. Run your fingers over its flat sides. They should be perfectly smooth. If not, it is dirty. Clean it free of dried gel. If it is not smooth, later when you set your clamps, you will break your glass panes. Give a smoothness check of a second spacer also.
4. Lay the spacer atop the sides each short edge of the pane. The spacer should protrude over the "top" by a millimeter or so.
5. Lay the second pane so that it bridges the spaces with it's "top" about even with the "top" of the lower pane. As the figure to the right shows, the spacers protrude a bit beyond the top.
6. Lift one edge of the "sandwich" and insert one of the side clamps. Lightly snug one of the middle screws only. (Don't loose track of the "top")
7. Install the second side clamp and again lightly snug its middle screw only.
8. Find a clean, and perfectly flat surface. Rub your fingers over the surface to assure yourself of its smoothness.
9. Hold the sandwich assembly vertically upside-down on that part of the countertop so that the "top"s of the panes are nearly at the surface of the countertop.
10. Loosen both middle screws of the two side-clamps, and slide the side-clamps so that their ends rest on the countertop.
11. The panes and the spacers will drop into place such that ALL these are in ONE plane - the tops of the clamps, the panes and the ends of the spacers. (The whole slightly-loose sandwich ought to be able to stand precariously on its own at this time.) It is VERY necessary that all these are in a single plane because later a rubber gasket must go flush across all these components to make a water-tight seal.
12. Inserting something thin (another spacer?) between the panes (figure to the right), push the spacer on one side into the clamp while holding that clamp and pushing it so it snug with the two panes of glass. When in place, snug up one of that clamp's screws. Do the same process with the other side.
13. Pick up the clamped sandwich and inspect the "top" - all should be properly aligned (planar). This is your last major quality-control step. If things are out-of-alignment, the device will later leak and be unusable. (The "bottom" will probably not be in perfect alignment. That is okay.
14. Lay the sandwich down, and snug up all the clamps' screws.
Racking the Glass Assembly
1. Find a solid rubber gasket - one that does NOT have a slot running for most of its length.
2. Lay it in the bottom of an assembly stand such that its grooves match those of the stand. Center it lengthwise.
3. Slip the glass sandwich with its clamps into the stand such that the "bottom" edges of the panes are downward and in the stand.
4. Insert, but do NOT tighten two eccentric screws through the stand's holes into the side-clamps.
5. You will probably notice that there are gaps in the juncture between the gasket and the gasket. These we shall now proceed to fill using molten agarose.
6. To 20 ml of cold glass-distilled water, add 200 mg of agarose (not agar!).* Heat (flame or microwave oven) with swirling to dissolve and melt the agarose.
7. Lift the glass assembly in the stand about 1 mm.
8. Using a plastic dropper, quickly transfer some of the agarose to where the bottom edges of the panes meet the rubber gasket. Make a good molten bead along both bottoms of the pane edges.
9. Quickly snug up the two eccentric screws. Be gentle! Do NOT over-tighten! If you do overtighten, you will cause the panes and spacers to shift in the clamps, and this will pull the "tops" out of proper alignment, which you will later need to be absolutely planar. Check the "tops" to make sure you haven't dislodged them. (If they have moved and are no longer exactly planar, "go back to GO! and do not collect...") You will likely see the molten agarose be pulled by capillary action between the glass bottoms and the gasket, and perhaps even into the space between the panes. Good! The snugging of the eccentric screws will assist in the agarose's making a water-tight seal all around the bottoms.
10. Within a couple of hours of doing the above (no longer as the agarose will dry and fracture), go to the page on using your time efficiently in making the gels. You will be making a polyacrylamide gel between the panes.
Loading your samples for assay.
1. Turn the sandwich in the stand upside down and allow the excess fluids and butanol to drain from the sandwich onto a paper towel.
2. Turn upright again, and now fill the slot with 1X-electrophoresis buffer. (400 ml glass-distilled water + 100 ml 5X-chamber buffer). The remainder of the solution will be used later.
3. In your notebook draw a diagram of your loading pattern. The first and last wells ofter result in skewed, "smiling" patterns so refrain from using those wells.
4. To a 50 μL or 100 μL pipetter attach a special long, narrow gel-loading tip or a fine-gauge, plastic-flanged hypodermic needle - a needle narrow enough to fit between the panes, and long enough to reach the wells.
5. Fully draw up 10 μL to 50 μL of the blue sample into the needle or tip.
6. Holding the pipetter vertically in one hand, and the shaft of the needle between thumb and index finger of the other hand. Using the "needle fingers" steady the insertion of the needle between the panes and push the needle down to about halfway into the desired well.
7. SLOWLY eject the blue solution, which is dense due to the high amount of glycerol, and will sink to the bottom of the well and stay there. At times a small bubble or two might be ejected from the needle. Because the needle tip is halfway up the well, it will escape without stirring the blue in the well. Do not fill the well more than 2/3 full. Withdraw the needle straight up. Any residual blue solution will viscously flow down into its specified well.
8. Do this for all of the samples. You will not lose track of where you are because the filled wells are conspicuously filled with prior samples.
9. When finished with the loading needle, rinse it with water and blow air through it to that it will dry unclogged.
Installing the Top Buffer Chamber
1. Hold the top buffer chamber upside-down in you hand. (Don't set it down upside-down because its electrical connection is liable to be broken.)
2. Insert slotted rubber gaskets into both sides making sure that the rubber ribs fit into the chamber's grooves, and that the slot in the rubber coincides with the slot in the chamber.
3. Using your fingertips to hold the gaskets in place, turn the chamber right-side up and slip it down over the sandwich.
4. Insert the four required eccentric screws. These may be stolen from the bottom where they are binding the sandwiches to the stand. Tighten the screws so that the lines on one arm are pointing directly at the gasket.
5. If everything was assembled correctly, you should be able to look straight down into the upper chamber and see the blue solutions in the sandwiches.
Filling the Chambers
1. Fill the bottom electrode chamber up to the line, which you hope someone has helpfully drawn for your convenience.
2. Insert the cooling device, which has a platinum electrode running along its bottom and its banana plug at the top.
3. Lift the upper chamber with attached sandwiches up out of the stand. Remove any agarose that clings to the bottoms of the sandwiches. Slide the assembly into the lower chamber. It will fit only one way so that both electrodes' banana plugs are close together.
4. CHECK FOR MASSIVE LEAKS. If you have done everything with gentle snugness, you should not have leaks. (Leaking is minimized in this apparatus because as you lower the top assembly into the bottom chamber, the buffer rises around the upper chamber to the height of the buffer you will want to put into the upper chamber. Leaks will cause an electrical short-circuit and slow your progress. The bigger the leak, the slower.
5. Dump the remainder of the electrode buffer into the top chamber being careful to not pour it directly into the slots. Doing so will stir up the wells below.
Electrical Connections
1. Set the top on the upper chamber and push the banana plugs up into their recepticals. Note that the black lead goes to the top electrode and the red to the bottom chamber. Black = negative; red = positive. Maintain this arrangement if your experimental substance has a net negative charge (LPS, nucleic acids, most proteins and bromophenyl blue tracking dye).
2. Plug the leads into your DC-voltage power supply maintaining black into black and red into red, if that is the arrangement you want.
3. Turn all the dials to zero before plugging the power supply into the wall.
4. For gels of about 15 cm length you will want about 100 to 120 volts-DC. (This is very handy because any homemade RECTIFIED power supply can feed directly off 110VAC house current. Amps will be dependent on the width and thickness of the gel.)
5. Turn up the voltage to about 100 VDC or to about 10 VDC/cm length of gel if your gels are shorter. (Higher voltage will mean more amps, and more amps means more heat is generated. As LPS is not heat sensitive, warmth doesn't matter and the cooling system need not be used. But you don't want the gel to begin boiling!
6. Observe!
  1. Very fine bubbles of hydrogen gas begin immediately rising from the upper chamber's platinum electrode. If they don't, but rise from the bottom chamber's electrode, pull the plug and reverse your wires, and replug. If no fine bubbles rise anywhere, then you don't have electricity flowing for some reason.
    1. Your upper gaskets were solid when they should have been slotted. Siphon out the upper buffer and detach the chamber from the sandwiches and install the correct, slotted gasket; reassemble.
    2. Your banana plugs or leads might be defective. Wiggle the connections and see if bubbles begin to rise. Or replace the lids and attached leads.
    3. Your power supply is defective. Do NOT try to spark the leads. These devices are made with very sensity current interrupters which will kick in if you touch one electrode to another. (This is to prevent you from getting a dangerous shock.) Instead get a voltmeter and check to make sure that there is truly a voltage across the electrodes.
    4. If the above are not the problem, then you must be living in the wrong phase of the moon! Quit and let your lab partner do it!
  2. Within a minute you should see your blue tracking dye begin to penetrate the stacking gel. If the dye is rising out of the wells, your connections are reversed.
The Electrophoresis
1. Every hour or so, look in on how the progress of the tracking dye is moving. A good run will soon have the tracking dye as a very thin line marching slowly down the gel.
2. While the gel is "running," prepare your developing solution(s) - particularly the first one.
3. You will note the "edge effect" as the tracking dye moves downwards. There is a "smile" at the edges.
4. When the dye is about a centimeter from the bottom, unplug the power supply (that is the surest way you know it is "off" and you won't get a shock).
5. Immediately do the next step.
Disassembly
1. Washing equipment must be exclusively for SDS-PAGE stuff as the technique is so sensitive that other grime will contaminate the components. So have handy its designated dishpan and sponges. You will not need detergent as there is so much SDS (the same stuff as in dishwashiing detergent) that additional is not needed.
2. Lift off the lid with its leads and place far from a sink. You don't want to get them wet or they won't work in the future.
3. Lift out the top chamber with attached sandwiches. Dump the used buffer into a sink.
4. For the following steps, be aware that SDS is slippery detergent!
5. Lay the assembly on its side, and detach one sandwich and then the other.
6. Once the sandwiches are removed from the top chamber, clean all the fragile parts of the upper and lower chambers and set them safely away to dry. Do it now! Any broken parts - no more electrophoresis in your future! These are expensive because of the platinum imbedded in them.
7. Remove the clamps from the sides of one sandwich.
8. Lay the sandwich on the countertop with the "top" overhanging the edge an inch or so.
9. Use a spare spacer: insert it a few mm into the top and push the side spacers outwards and remove them. Drop them into a water-filled dishpan in the sink.
10. Still with the glass sandwich slightly overhanging the countertop edge: Using a thin plastic wedge (not metal!), slowly jiggle it into the top of the sandwich near one corner. (Often the handles of plastic picnic knives make good wedges.) With one hand holding the far side of the sandwich and with a thumb or palm on the glass, push and jiggle the wedge using your other hand, but do not pry. If all goes right, the panes with separate. (If all goes wrong, the pane will break and your pushing hand will only be able to move forward to the edge of the countertop and not get cut on the glass. See, every little item listed here can be important!)
11. Once the panes have separated, you will probably find for some anti-gravitational reason that the gel is stuck to the upper pane. If so, pull out the wedge and turn the sandwich over, and use the wedge again to easily lift off the top pane.
12. Set the released pane in the dishpan (remember how careful you must be not to chip the long edges!).
13. Using a knife, cut away any agarose at the bottom of the gel, and also all but a little of the well-crenelations at the top of the gel. Using the flat edge of a spare spacer, push and slide the cut-offs directly into the wastebasket.
14. Open your first developing solution container. (See SilverStaining)
15. Holding onto the top edge of the pane, invert it over the receiving container - just an inch or two over the liquid - and using the wedge, slip it under one of the BOTTOM corners of the gel. The gel will peel itself away from the pane and fall into the solution.
16. Slosh the container a little to thoroughly wet the gel.
17. Put the pan into the dishpan and attack the second sandwich.
18. Put the second gel into the same receiving container, and slosh.
19. It is very important that the two gels remain in sloshing mode from here on. Otherwise, they print upon each other.
20. Get your various developing solutions lined up for SilverStaining.
21. Using a coarse sponge clean the panes and spacers. Rinse in distilled water and stand up to dry.
22. Proceed with developing the gels.

*agar consists of approximately 5 different polysaccharides. Four of these have sulfates and phosphate side-groups and thus are electrophoretically mobile. Agarose is the remaining, uncharged component.