Jacobs:Protocol Agarose Formaldehyde Gel Preparation for RNA Electrophoresis

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Contents

Materials

  • Agarose
  • Formaldehyde
  • 10X MOPS buffer
  • RNAse free water
  • Ethidium Bromide
  • Glass bottle
  • RNA Sample
  • Microwave
  • Agarose gel electrophoresis system (gel box, casting tray, lid, power supply)
  • Blue Juice dye, 10x
  • RNA ladder
  • Ice bucket
  • Ice
  • Serological pipets
  • Pipet aid
  • Pipetter
  • Filtered pipet tips
  • RNAse free microfuge tube
  • UV transilluminator
  • Camera
  • Ethidium bromide extractor


Procedure

  1. One agarose-formaldehyde gel will be prepared for the class by one person
  2. In fume hood, add 1.95g agarose, 108.23ml RNAse free water, 13ml 10X MOPS in a 500 ml glass beaker (this makes 1.5% agarose gel solution)
  3. Heat until solution is clear and boiling in the microwave, it will take approximately 1 min for the agarose to dissolve completely (*Note: Be sure to loosen the cap to the bottle before heating the solution in the microwave. Use a large volume glass bottle and observe the bottle during heating to prevent overflow of the solution as it begins to boil.)
  4. In fume hood, measure 8.78ml formaldehyde using a graduate cylinder
  5. Slowly add formaldehyde to the agarose solution by slowly pouring down it down the side of the beaker, do not create bubbles
  6. While still in the fume hood, add 6.5μL of ethidium bromide (final concentration 0.5μg/mL)
  7. Let the agarose solution cool for 5 minutes
  8. Place gel tray in electrophoresis apparatus making sure that the entire tray is level and that the red gaskets on the edges of the gel tray run lengthwise along the edge of the apparatus, the red gaskets should form a complete seal around the casting tray within the gel box
  9. Place the 20-well gel comb into the slots of the gel tray
  10. Pour the cooled molten agarose (50-60C) solution into the casting tray
  11. Allow gel to solidify at room temperature, 20-40mins, the agarose gel will be white in color when it has solidified 2
  12. While the gel is solidifying, make 700mL of the running buffer. Dilute 10X MOPS to 1X MOPS by mixing 70mL 10X MOPS with 630mL of RNAse free water. Add 35μL of ethidium bromide (final concentration 0.5μg/mL)
  13. While gel is solidifying, add the blue dye (10X) to the RNA sample volume such that the final concentration of Blue Juice dye is 1X. For a 20μL sample, 2μL of blue juice is sufficient. Keep RNA sample with blue dye on ice.
  14. Remove the gel tray from the casting tray and rotate it 90 degrees so that the red gaskets now run in the perpendicular direction within the electrophoresis apparatus, the loading wells must be on the black cathode (-) end of the apparatus, the RNA samples (highly negatively charged) should always run towards the red anode (+)
  15. When the agarose gel has solidified, carefully remove the comb from the solidified gel
  16. Gently pour the 1X MOPS over the gel until it is submerged under at least 1mm of the running buffer
  17. Load 25 ul your RNA sample into the wells using a Pipetman 200 and filtered pipet tips
  18. Add 15 μL of the DNA ladder into the top well
  19. Gently place lid on apparatus
  20. Run the gel at 100V for about 1.5 hr (run time may vary depending on gel composition and thickness)
  21. Ensure that the blue dye front is running towards the red anode (+)
  22. Stop the gel before the blue dye front runs off of the gel
  23. Place gel on UV transilluminator for visualization of the RNA bands 80-85% of RNA isolated from cells is ribosomal RNA (rRNA) (28S, 18S, 5.8S and 5S), 15-20% is transfer RNA (tRNA) and 1-5% is messenger RNA (mRNA); therefore, your sample will be mostly rRNA with bands at 5kb and 2kb.
  24. Run excess MOPS buffer through an ethidium bromide extractor before disposing of it down the sink
  25. Log the volume of buffer run through the ethidium bromide extractor and wrap in plastic for storage


References

Contact

  • History: CRJ-ABC, Last updated 8/15/07


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