MIT iGEM Agarose Gels

Running an Agarose Gel

Brief intro:
Agarose gels are analytical and functional tools for physical partitioning of DNA fragments. The DNA fragments are separated by size on the gel and are visualized by fluorescent dyes or ethidium bromide. The visualization of the DNA in the gel allows us to analyze restriction digests and the presence/absence of correct PCR product, among other things. We can also cut out desired fragments of DNA that have been separated from other DNA (such as a plasmid w/ an undesirable antibiotic marker) for further cloning.

Materials

• DNA, the thing you want to analyze/cut out. Measure concentration in nanodrop beforehand.
• 1xTAE buffer. Make a 1/10 dilution from bought iGEM stock.
• SybrSafe dye, 10,000x. Use iGEM aliquot.
• Gel well and comb. Use appropriate comb for your need. For analytical gels, can use thin combs. For preparative gels, use wide-toothed combs.
• Melted 1%Agarose in 1xTAE.
  Prepare ahead of time by melting appropriate amount of Agarose mixed in TAE in the microwave. (eg. to prepare 500ml of melted 1% agarose, mix 5g of agarose in ~495ml 1xTAE in a foil-covered flask and microwave for 6+min; keep an eye on it every couple of minutes when you microwave so it doesn't boil over; be careful to wear gloves when handling hot liquid containers). Can keep the melted agarose in the 65*C oven for convenience or leave on bench and re-melt every time you need it.

Setting up and Running the Gel

1. Set up your clean gel tray by taping both sides w/ lab tape and putting the clean comb in place.
   Thin-toothed combs will hold <20µl.
   Wide-toothed combs will hold <50µl.
2. Measure out the needed amount of melted agarose in a cylinder and pour it in a beaker.
   For small gels, use 35-40ml.
   For large gels, use 95-100ml.
3. Add appropriate amount of 10,000xSybrSafe dye to the melted agarose. (eg. for 35ml of agarose, add 3.5µl of SybrSafe).
4. MIX the SybrSafe into the agarose by swirling the agarose.
5. POUR the agarose into the gel tray you prepared.
6. WAIT ~10min for the agarose to harden. It should turn a somewhat opaque light blue.
7. While waiting for the agarose to harden, MIX your DNA sample w/ 5x Ficoll Orange stain. (eg. for 10µl of PCR product, add 2-3µl of 5x Ficol Orange stain). The stain has dense glycerol in it and makes the DNA fall to the bottom of the well.
   You don't need to stain the ladder DNA, since it's been pre-stained.
8. Once the agarose has hardened, REMOVE the tape and PLACE the gel in the gel box. Make sure the gel is covered in 1xTAE buffer.
9. Remove the comb GENTLY! This is easiest by pulling the comb up from one side first; don't try to pull the whole thing out at once or it'll break.
10. Load your DNA. 500ng of the ladder is usually plenty. If you can't see the wells, try placing a dark blue nitrile glove behind the gel box to increase contrast between the empty wells and the rest of the gel.
11. Hook up the leads to the correct plugs and turn on the power supply.
12. Typically, we run a small agarose gel at 100volts for 1hr, but you can vary the time and voltage according to your time/resolution needs.
    Be careful not to let the current become >110mA or the gel might start to melt. You can watch the Ficol Orange stain travel ahead of the DNA, so once it reaches the end of the gel you know you can't run it any longer.

Viewing the Gel

1. Turn OFF the generator and power supply.
2. Using gloves, REMOVE the gel tray from the gel box. BE CAREFUL! Don't let it slip from the tray and break! Use both hands.
3. Bring the gel to the Endylab gel box in the supply room. Wipe down the viewing tray in the box to remove residual Ethidium Bromide that may have been left by the Sauer lab.
4. Place the gel on the viewing tray (can be left on the gel tray, but the image can be improved by removing the gel from the tray), slide it into the box, and close the sliding cover.
5. Open the GeneSnap software if it's not open already. Make sure the top left button is green. If it's red, click on it to freeze the image.
6. Set exposure time to 150ms, light source to Upper White, filter to No Filter. Click on the Green button in the upper left. You should now be able to view your gel in white light and adjust its position and the zoom and focus w/ which you view it.
7. Once you're happy w/ your gel's position/zoom/resolution, click on the red button in the upper left. This freezes the image. Now, change exposure time to 2s, light source to Transilluminator, filter to Short Band Path (this might take a couple of tries to click before the sofware accepts it; just keep clicking it).
8. Click on the green button. Your gel should appear as seen through UV light. If it looks OK, IMMEDIATELY PRESS THE RED BUTTON. If you leave the transilluminator on, the dye bound to your DNA will bleach and you won't be able to re-image the gel if you need to. Also, UV damages DNA, so if you need to cut out your DNA, you might be incurring damage.
9. If you are not happy w/ your gel image, re-adjust the settings and re-image until you are happy. Sometimes simply playing w/ the exposure time can make a hard-to-see band easier to visualize.

Cutting out Gel Bands

1. Once you've imaged your gel in GeneSnap and have confirmed that the band you want to cut out is there, bring your gel to the UV lightbox next to the Gelbox setup.
2. Remove your gel from the tray and place it on the UV lightbox.
3. Be sure you have on hand a sterile razor blade and empty epp-tubes into which you can place your gel slices.
4. Don the appropriate protective gear (lab coat, eyewear) and close the door to protect yourself and other people from the UV.
5. Turn off the light in the room and turn on the UV lightbox using the knob on the front left of the box.
   One click clockwise goes to Analytical, which is very bright and damages you and the DNA; another click goes to Preparative, which is less bright; try to use the Preparative setting if you can.
6. Cut out the desired bands carefully using the razor blade. Work swiftly to minimize the amount of time the UV light is on and therefore the amount of damage to the DNA.
7. Once you are done w/ your gel, gather up the pieces and dispose of it in the hazardous waste bin labeled "SybrGold".
8. Use the Gel Extraction protocol from Qiagen to extract the DNA from your gel. Expect low yields. This kit at