Agarose Gels: making, loading, and imaging

Note: Avoid skin contact with Ethidium Bromide and UV exposure.

Making the Gel

  1. Secure the gel tray on the loading block.

  2. Measure 100 mL of 1X TAE buffer in a graduated cylinder (if making thick gel, 200 mL 1X TAE).

  3. Measure out agarose to intended percentage using the analytical balance. (1% gel: 1.0 g / 100 mL, 2% gel: 2.0 g / 100 mL)

  4. Add agarose and 1X TAE to an Erlenmeyer flask and mix by gently swirling. Microwave for ~2 min. and monitor for boil-over.

  5. When the mixture starts to boil, stop microwave, remove flask using hot pad, and slightly swirl mixture.

  6. Replace in microwave and continue microwaving until mixture comes back to boil (~5 s.). Remove flask from microwave using hot pads, slightly swirl once more. All agarose should be dissolved.

  7. Place on bench-top and monitor the temperature using a thermometer (located in gel rig area).

  8. Once the mixture has reached ~70ºC, add 10 μL of Ethidium bromide and swirl.

  9. Pour the agarose into gel mold slowly to avoid bubbles. Place appropriate comb and pop any bubbles in the gel using a pipette tip. Allow to cool until solid (15-30 min. for a 100 mL gel).

  10. Once solid, remove gel tray from loading block and place in the gel rig (orient the wells so that they are at the negative electrode). Black is negative, Red is positive. Run to Red.

  11. Fill gel rig to the max line with 1X TAE buffer. Carefully remove comb, so as not to destroy any wells.

Loading and Running the Gel

  1. Load 10 μL of DNA ladder into well(s) furthest to left.

  2. Add 5 µL of one tube of PCR product into a well. Repeat this until all PCR product has its own well. Make sure to note where you placed which PCR product in which well.

  3. Mix PCR product with loading dye prior to placement into gel well, if not a component of the PCR reaction.

  4. Always store leftover nucleic acids at 4ºC until gel is finished running in case the gel fails to work.

  5. Run gel at 80-150V for 1-1.5 hr. or until dye line is 75% down the gel.

Imaging the Gel

  1. Bring the gel and gel tray to the AlphaImager HP.

  2. Open the door to the imager and slide gel onto the black stage.

  3. Close door. Put the filter wheel on #2 for ethidium bromide.

  4. Select the Transillumination UV button in the top right corner of the imager.

  5. On the computer, open the AlphaImagerHP application.

  6. Once the main application screen is up, select Acquire.

  7. Click the Expose Preview button (green box), and the gel should appear on the screen. If not, open the door of the imager and move the gel, so that you can see it on the screen. Set the exposure time to a value that gives you the best image.

  8. Once you have the gel in frame and it is to your liking, click Acquire Image (red box). Wait a few seconds for it to take the image.

  9. Once the image appears, click on Image (along the top toolbar) then click Conversion, and then click 8-bit grayscale.

  10. To save the image, click Save As (under File in the top toolbar). Select Zamanian Lab. Under the Zamanian Lab folder select your folder. If you do not have a folder, make yourself one by right clicking and selecting New Folder. Name the folder with your name. Click the newly created folder. Once in your folder, name the file in the format of YYYYMMDD.jpg and click save.

  11. Save the image to a flash drive. If available, print the gel on thermal paper by clicking File, then Print.

  12. Once image is saved and printed, close out of all windows, remove gel from the imager, turn the UV light off, dump the gel in the biohazard trash, and upload gel to Box and place into Benchling.