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Agarose Gel Electrophoresis

Theory

After performing 16S PCR, one of the best ways to determine success of the reaction is to run your amplicons through a gel in gel electrophoresis. Gel electrophoresis separates out your samples based on size (number of base pairs). This can be compared to a known ladder to determine the sizing of your amplicons and make sure they are correct.

Prepare Your Workspace

  1. Turn on PCR Clean Hood blower and white light.
  2. Clean the hood space with 70% ethanol including the work surface and walls.
  3. Turn off the white light and turn on the UV light. Make sure you turn the dial past 10 at minimum. ⚠️ Do not trust the glass to protect you from UV exposure. You can be in a different part of the lab while it is running, but do not loiter in front of the cabinet.
  4. When the UV light turns off, turn on the white light. You are ready to begin.

make sure to clean all materials with 70% ethanol before putting them into the cabinet

Gather Materials

  • Agarose Powder (or prepared 2% gel)
  • 50X TAE Buffer
  • PCR Amplicons
  • Micropipettes (P10, P20)
  • Pipette Tips (10uL, 20uL)
  • 100bp DNA Ladder
  • 6X Loading Dye
  • Sybr Safe DNA Stain (ThermoFisher Scientific, S33102)
  • Graduated Cylinder
  • Erlenmeyer Flask
  • Electrophoresis Cell and Power Box
  • Gel Casting Tray
  • Gel Combs (20- or 30-wells)

Make a Gel

  1. Make 1X TAE Buffer by mixing 20mL of 50X TAE Buffer with 980mL of Distilled Water.
  2. Mix 4g Agarose with 200mL of CLEAN 1X TAE Buffer.
  3. Melt in microwave for 30 seconds, remove and swirl to mix. Melt for another 1-2 minutes until completely melted and dissolved.
  4. Allow gel to cool to 55dC.
  5. Add 20uL of SYBR Safe DNA Gel Stain and swirl to mix.
  6. Assemble the gel mold by placing the gel try in the mold and tightening into place. Put the desired number and size of combs into the mold. This depends on the number of samples to be run and the size of the gel.
  7. Pour the melted gel into the mold and let set for about 30 minutes.
  8. Once set, pull the comb straight up to remove. Be careful not to damage any wells.

Run the Gel

  1. Place the clear gel mold containing the gel into the electrophoresis cell, ensuring that the wells are on the side with the black electrode as DNA runs toward postiive.
  2. Cover with clean 1X TAE Buffer. Ensure that all wells are completely covered but that you are not above the max fill line.
  3. Make the DNA ladder by mixing 1uL of 100bp DNA Ladder, 1uL 6X Loading Dye, and 4uL Nuclease Free Water. If there are multiple rows, multiply each reagent by the number of rows.
  4. Load 3-5uL of DNA Ladder into the first well of each row.
  5. Add 3uL of 6X Loading Dye to each of your PCR samples.
  6. Load 3-5uL of PCR sample into the remaining wells until the gel is full or there are no samples left.
  7. Place lid onto the electrophoresis cell and turn it on.
  8. Set the voltage at 150V and the time to 45 minutes.
  9. Click 'Run.'
  10. When the gel is finished running, view on the ChemiDoc (owned by the Ott lab) in room 321C to determine the results.

Viewing the Gel

  1. In room 321C, there is a ChemiDoc. We use this for viewing the gels and saving the output image.
  2. Sign the logbook by the computer.
  3. Sign into the computer.
  4. Open the ImageLab software.
  5. Open the door to the ChemiDoc and pull out the sliding drawer. Place your gel on the glass and make sure to remove any large air bubbles.
  6. Slide the tray into the machine.
  7. On the program, select 'New.'
  8. In the top section, using the dropdown menu, select 'Nucleic Acid Gels' and 'SYBR Safe.'
  9. Click 'Position Gel' on the left side of the screen.
  10. Make sure that the filter is on 'Filter 1.'
  11. Zoom out so that you can see the entire gel and line it up so that it is straight.
  12. Once you have the gel in a position that will work, close the door firmly.
  13. Click 'Run.'
  14. The program will output an image of your gel on the screen. You can adjust the contrast to make bands easier to see or less bright.
  15. Under 'File' select 'Export' and 'Export for Publication.'
  16. You can then save the image as whatever you'd like - make sure to set the filetype as .jpg instead of the default .tif file ❗
  17. Upload your image to your OneDrive using the Google Chrome or Microsoft Edge browser.
  18. Close all programs and sign out of the computer.
  19. Pull out the tray and remove your gel. Spray wiht 70% ethanol and wipe down with a KimWipe.

Troubleshooting Your Gel