Introduction: DIY Gel Electrophoresis Box for Separating DNA
Making a gel electrophoresis set-up is conceptually quite simple. A tank with a tray with two naked wires at either end of the tank though which current flows. The tray contains a gel made out of agarose which is placed inside the tank and filled with an electrically conductive buffer (water plus salts). The molecules (DNA, RNA, proteins, dyes) move under the force of the electrical voltage. Smaller guys and guys with more charge (and more pizzaz?) move faster. So you get a nice separation.
I will be presenting three instructables:
- How to make the electrophoresis box (this one)
- How to make a simple electrophoresis power supply - a trick with this power supply is that it virtually extends the length of the gel. The longer the molecules travel the better the size resolution.
- How to use DNA electrophoresis for some simple experiments, like paternity testing!
The simplest way is to modify an existing plastic or glass box for the external reservoir, and a smaller plastic box for the gel tray but I had imposed stupid constraints on my set-up like - it should be about 6 inches square (just because most of my other projects are 6 inches ...), it should use up materials I have been collecting and that were gathering dust, and it should be as good as professional units that cost hundreds of dollars.
My requirements were
- Strong box that should last years, so not flimsy foam core boards and duct tape
- Ability to cast the electrophoresis gel in the gel box itself, no additional casting trays, etc.
- Flexibility in number of samples I can load and run, that is, combs (that create holes in the gels for sample to be loaded) should be in different sizes and formats
- Removable electrode holders. If I want to play with different types of electrode materials or I mess up the electrodes, and
- Everything in less than 6 inches x 6 inches
The final product is shown in the first picture plus the design mock ups in Sketchup are shown in the next three images. The first Sketchup shows all the parts of the gel box. The two electrode holders on the left, the electrophoresis tank in the middle with the gel tray behind it, and two sets of combs on the right. The 2nd Sketchup shows the electrode holders and gel tray placed inside the tank. And the last shows the combs inside the gel tray.
A youtube video summarizes the construction steps.
Step 1: Making the Agarose Gel Tray
The gel tray is a U-shaped tray in which the agarose will be poured. Agarose is like gelatin, on heating it becomes liquid and when it cools it becomes solid again. The trick here was to make the tray square so that it fits in the tank in two ways, with the open ends of the trays facing the electrodes so current can pass through the gel or rotated 90 degrees so that the open ends are then blocked by the walls of the tank forming a dam to cast the gel.
The 2nd image shows the parts for the gel tray as well as the base for the tank. The gel tray is 4 inches square. The sides are 1.75 inches tall.
Had not worked with acrylic (plexiglass) before but the plastic was cut easily with a circular saw in which I had put a cheap steel blade which did not have carbide tipped teeth. Ran it slow. The edges produced were not too bad. I cut 1 cm deep slots into the two sides of the tray for the comb holders on the band saw.
I was not sure how to glue the pieces together so tried a few solvents I had at home on scrap pieces. The dollar store nail polish remover did not work. Acetone worked well but it evaporates so fast that you end up consuming a lot in no time. The 'Goof Off' cleaner that I had bought from Harbor Freight worked really well. It was quick at joining but did not evaporate quickly and gave me a few seconds to readjust parts if required. I put tiny drops to hold the parts together first, a bit like spot welding, and then placed a small amount to be sucked in by capillary action into a joint. After a few minutes the two parts were fused together.
I cut a slot into the sides, you can barely see these in the last picture. The slots will be for a rubber gasket that will hold the tray tightly in the tank when pouring agarose.
Step 2: Making the Outer Electrophoresis Box
To make the base, I cut two 2 inch x 6 inch pieces from acrylic, cut slots in the side to hold the electrode holders and then fused 2x6 inch pieces to the 6 inch square base approximately 4.1 inches apart (used the gel tray that I just made to measure the gap plus added bit for the tray gasket). I then fused a 4.1 inch acrylic piece to form the third wall for the tank. I did a water test to make sure that there were no leaks. None. So went ahead and added the fourth wall. Did the leak test again. Unfortunately there was a slow but steady drip of drops from the the two corners.
Step 3: Fixing Leaks in the Electrophoresis Box
Did a google on how to fix leaks in plexiglass tanks. Found a lot of comments from folks who own fish tanks - most said you were screwed, only way of fixing was fusing a new vertical strip of plexiglass butting against the joints.
I thought I would try making a gooey form of plexiglass by dissolving as much of the plastic in the Goof Off solvent. I put the small pieces and dust generated from sawing the acrylic in a Starbucks Frappuccino bottle with the solvent and let it soak overnight with the lid tightly on. Next morning all the small pieces had dissolved and the liquidy solvent had turned more syrupy. I added the syrup to the leaking inside seams of the tank and within a few minutes, the leaks were gone! Anyone want to market this as a acrylic tank leak fixer, please let me know.
I then added four tiny vertical pieces of acrylic to the long sides of the tank to keep the gel tray from sliding when it is placed inside the tank.
Step 4: Making the Electrode Holders
For the electrode holders, I cut two pieces that were about 4.5 inches wide by about 2 inches. The actual sizes were taken from the tank dimensions. I cut out two vertical pieces from the sides of these two plates leaving a 1 cm shoulder which would fit into the corresponding slots in the tank. I then cut holes for the electrical sockets in a scrap piece of acrylic and then cut out the L-ish shaped pieces on the band saw. One L-shaped piece was fused to each electrode plate. I added a small scrap of acrylic connecting the L-shaped piece to the electrode plate for reinforcement. The electrical female sockets were also glued in place with my acrylic syrup.
I had really thin platinum wire from an organic chemistry lab that I had done decades ago. Had not touched these since then. I used a hot needle to poke two holes in each electrode plate for the platinum wire. I stuck one end of a piece of this really thin and, am guessing, really expensive wire into the tiny hole at the far end and then put a drop of my acrylic syrup that I had prepared to seal leaks. I threaded the wire into the other hole and pulled it through till it reached the electrical plug socket and snipped it off. I put a piece of thin plastic tubing over this wire and then soldered the wire to the plug socket. Checked with an ohmmeter just to make sure.
The plastic tubing is to make sure that the electrical current only flows out from the exposed horizontal wire and not the plastic encased vertical wire.
The exposed connection between the platinum wire and the socket was covered with plastic end caps that I 'harvested' from ballpoint pens. I cut a slot in these caps so that the cap would slide over the thin plastic tubing, slid them over the exposed soldered joints and filled the caps with hot glue.
For those who do not have access to platinum wire, stainless steel or monel wires (from boating supply stores) can be used. Iron ions tend to cleave DNA so does Zinc and Lead so caution here. The electrode plates can also be covered with graphite. A good source is spongy graphite coated filters used in air purifiers. Or use thick pencil leads. Have not tried this but worth a shot.
Step 5: Making the Gel Combs
For the gel combs, I decided to create two sets that would fit into the slots cut into the gel tray. Two horizontal cross pieces were cut and tiny pieces fused close to the ends so that the cross bars would not slide.
For the combs, I needed the fingers to be at 9 mm spacing. This weird number is the standard spacing used in microplates and multichannel pipettes. I decided to make a two sided comb, one with 8 fingers that were the required 9 mm spacing and one side with 16 fingers that had 4.5 mm spacing. I had already decided on this configuration when designing the gel tray so the gel tray was wide enough to take these wells.
When looking around for materials to make the comb from I came across a box for a Skagen watch that my wife had saved. I decided to repurpose it for the combs as they were the perfect width for the combs and I could cut the two sets of combs in one go. I poured some water in this Skagen box, placed in a microwave and slowly heated it to higher and higher temperatures till boiling to make sure that the plastic material could handle the heat. The plastic did not even soften with water boiling inside. The heat resistance is required as hot agarose would be added to the gel tray into which the combs would be placed.
I drew a template in Xara drawing software, printed and glued onto the Skagen box and then cut the slots on the bandsaw. Cleaned up the slots with a files and finally ended up with two sets of combs.
The combs were attached to the acrylic cross bar with nylon screws so that could be adjusted or replaced with other comb designs.
Step 6: The Gel Box - Finished
I put some rubber tubing as gasket and poured a gelatin gel in the gel tray with a set of combs in the middle slot. The whole gel box was placed in the refrigerator for the gelatin to solidify. The first image shows the gel tray with a 3% gelatin gel in salt water with the comb still attached. The next image shows the comb removed with the nice array of wells.
I flipped the tray around in the tank, poured ice cold salt water into the tank. I loaded the wells with rose syrup (appears orangish in image but was red) and the rose syrup mixed with black fabric dye. The thick syrup drops into the wells. I applied some power (20V < 0.2A) to the gel and immediately became aware of a major screw-up. Electrolysis of salt water produces chlorine gas. You can see the fine bubbles (looks like froth) in one of the images. Turned on the fan to blast the chlorine gas away but thought it would be wise to stop this little trial after about 10 mins. The dyes were beginning to separate nicely though.
Thought would publish this so other folks can start messing around with the DIY electrophoresis set-up.
More to come - including a neat electrophoresis power supply.