Overview: Gel Electrophoresis is a process performed in order to visualize DNA samples and verify the success of PCR (TS PCR, Index PCR, or other) amplification. The gel is set up with melted agarose in a plastic molding container and inserted combs in order to form a mold with slots for the DNA samples to be inserted into. Then, a loading dye/gel red master mix is created and mixed with our DNA samples from our PCR plate. We then fill the mold with a TBE buffer to ensure the electric current flows through the gel and nucleic acids are able to move through the gel. We are now able to load the slots we created in the gel with the mix of loading dye/gel red master mix and our samples we created earlier. These samples are compared with a ladder of DNA molecules of a known length that allows u to estimate the size of DNA fragments. Lastly, DNA is negatively charged so by setting up the gel to an electric current, the samples will migrate towards the positive current and create a visualization of the DNA bands for us. The final visualization of the samples are seen through UV light.
- Clean workspace: Put on gloves and wipe down your work bench with 10% bleach solution and/or ethanol.
- Melt agarose for gel casting: Find a 400 mL beaker from the sink/drying rack or bench and two 50 mL falcon tubes of pre-made agarose from the gel fridge. Also get a metal spatula thing (above the gel bench or in the drawers). Use the metal spatula to dig the agarose out of the falcon tubes and into the beaker. Microwave the beaker with the agarose for ~90s. You want the agarose to be completely melted and just boiled- it should be clear. Remove the beaker from the microwave using the mitts on top of the microwave. Let the beaker with the agarose sit for ~1 minute to cool a bit. Use spatula to remove thin film that may have formed on the surface of the melted agarose.
- Set up gel boxes:
- Insert red rubber seals into channels on the sides of the gel trays (alternatively, you can take the edges of the tray)
- Insert the trays into the gel boxed with the red rubber sides flush against the walls of the box. Make sure the tray is in the box securely and level
- Insert 4 combs, each with 25 wells into the gel tray slots. Make sure the comb wells are centered across the tray
- Cast gel: carefully pour agarose into mold and leave undisturbed for at least 30 minutes to set.
- 1:1 loading dye/gel red master mix: Remove sufficient volume of 1:1 gel red/LD from gel fridge (vortex and spin).
- If there is not enough premade: Gel red (1:1600, not stock) and 6x loading dye (blue or green/orange) are stored in the gel fridge. They are light-sensitive so make sure you only pull out what you need and close the boxes and put them back right away.
- Based on your # of samples, figure out how much gel red (1:1600) and loading dye you need. You’ll need 2.5 uL of gel red and 2.5 uL of loading dye per sample, plus 4 ladder wells at the end of each row, plus an extra count just in case.
- Ex: for 96 samples, you’ll need 101 portions of the dye/gel red mix, 96 for your samples + 4 for ladders + 1 for extra. So that’s 252.5 uL of loading dye + 252 uL of gel red (101 * 2.5 each).
- Get a clean 1.5mL tube and add 253 uL of loading dye and 253 uL of gel (it’s okay to round since the p1000 isn’t precise enough for decimals, they just need to be equal amounts). Feel free to make extra, especially if you know you will be running multiple gels that day/week.vortex and spin.
- Dispense 1:1 mix: Get a disposable/reusable USAscientific unskirted well plate (above the gel bench) and set it in a plate rack. Dispense 5uL of the gel dye master mix you just made into each well of the well plate that will have a sample.
- Options: use repeater pipette, regular micropipette to individually dispense dye, or use a multi-channel micropipette with the 1:1 dye in strip tubes.
- Vortex and spin samples: Retrieve your samples (in their well plates) from the freezer or thermocycler, defrost them, vortex them, and then spin them down. Use the big centrifuge next to the sink and make sure you balance your plate if you’re only putting one in (you can weigh it on the scale and try to get a balance that’s within a few grams of its mass).
- Add samples: Carefully peel the lid off your well plate with your samples in it. Use p10 multi-channel pipette to transfer 1 uL of each sample from your TS PCR plate into your other plate with the dye mix. Make sure you check that there’s actually DNA/volume in the pipette tips each time you transfer by holding tips up to the light. Repeat for columns 2-12, changing tips each time. Seal the dye+DNA plate with a temporary lid (silicone top ideally, can also use plastic or aluminum seal top) and vortex and spin it.
- Re-seal your TS PCR DNA sample plate with a fresh foil/clear plate seal, briefly label, and put your samples back in the freezer.
- Prepare the gel and buffer: The agarose gel should have set by now and look solid and semi-opaque. Pull the combs straight up out of the gel. Pull out the gel tray and take the red rubber stoppers off. Rotate the gel tray in the gel box so that the wells of the gel are closest to the black (-) node on the gel box. Once the gel tray is in place, fill the gel box with 1x TBE buffer (make sure it’s TBE buffer, not TAE) to just cover the gel (~600mL or up to the fill line on the gel box). Now you’re ready to load the gel!
- Loading the gel: Change the volume on your 12-channel multi-channel pipette to ~6 uL (which is the volume of dye + DNA). Using 12 tips transfer the volume in your dye + DNA well plate into the gel wells. Make sure you line things up correctly:
- There are 4 rows in the gel. You have 8 rows of sample + dye. They get interspersed, so that the four gel rows are AB, CD, EF, and GH.
- For the first transfer, you’ll take up 6 uL of row A from your well plate with 12 tips and dispense them left-aligned into the gel wells, so that the leftmost well is filled.
- Then, you don’t need to change tips, and insert row B in the wells interspersed between the ones you filled with row A. So it’ll go A1, B1, A2, B2, A3, B3, etc etc. There will be an empty well at the end for the ladder.
- Rinse tips in the buffer between rows by pumping up and down. Consider changing tips between each row, or at least halfway through the plate.
- Prepare your DNA ladder for the ends of each row:
- Cut a piece of Parafilm and set it on your bench (tip: tape to bench to it stays put).
- Dispense 5uL of the 1:1 loading dye/gel red mix onto the parafilm, creating 4 separate dots using the same tip.
- Then, add 1.5 uL of 100 bp DNA ladder (use aliquot) to each dot, mixing it up and down with the pipette tip (change tips for each dot).
- Load the ladder: Set a p10 pipette to 7 uL and dispense each dot into the empty well at the end of each of the 4 gel rows (can use same tip).
- Close box: Slide the lid onto the gel box. Make sure you line up red to red and black to black, with the wires, nodes, and ultimately into the voltage box.
- Run gel: Turn on the gel machine and run it at 120 volts for 30 minutes. Check when it first turns on to make sure that there are little tiny bubbles coming up from the bottom and the samples are being run in the correct direction.
- After 30 minutes, check to make sure that the faster/darker dye is more than halfway down the run, but not too close to the next row of wells. Run the gel a bit longer if you need to. But don’t run the fast dye off the gel or into the next row of wells.
- Clean up and image gel: Once your gel is done running, you can pour the buffer into the sink, or save it for later in the flask, and take your beautiful gel to the gel imager machine. Make sure to label gel so you can identify it later. Gel can be disposed in regular trash after imaging. Make sure you bring a flash drive so that you can transfer your gel photos.
See here How to Read a Gel Image