Samuel J Gurr, PhD

Gel Electrophoresis with ethidium bromide

by Samuel J Gurr PhD 4 min read

Gel electrophoresis (ethidium bromide)

Note: this protocol if you are imaging a gel today, turn on the computer for the GenSys software (computer takes a while to boot up)

Location: Room 18 at Milford lab Building 1

Materials (chemical):

  • ethidium bromide (aq solultion)

  • agarose I

  • 10x TBE

  • 1x TBE

  • DI water

  • DNA ladder

  • Gel dye

  • Extracted DNA samples (at least 5 ul per sample)

Materials (non-chemical):

  • 1 L and 250 ml erlenmeyer flask

  • gel tray

  • weighing paper

  • microwave

  • clock/timer

  • appropriate pipettes and tips

  • funnel for 1 L erlenmeyer

  • designated space for ethidium bromide (gels run here)

  • GeneSys transilluminator instrument and software

  • proper labelled waster containers for solid and liquid containing ethidium bromide

Protocol:

  1. For single small box, make a 50mL gel mix

  2. Making gels:

Note – lower agarose increases the size of pores that the DNA is traveling to resolve larger bands. Thus percent agarose is used for resolution based on target bands of interest. High percentage agarose increases separation/resolution of smaller molecular-weight bands (low bp) whereas low percentage agarose increases the resolution of higher molecular weight bands (i.e. 6% agarose gel for 10^4 – 10^6 bands).

For a 1% gel:

• small box: 50mL new 1X TBE (or TAE) buffer and 0.5g agarose

For a 1.5% gel:

• small box: 50mL new 1X TBE (or TAE) buffer and 0.75g agarose

For a 2.5% gel:

• small box: 50mL new 1X TBE (or TAE) buffer and 1.25g agarose

  1. Measure for your agarose solution:

  • Use a weigh boat to measure agarose

  • Use a 250 mL Erlenmeyer flask measure the TBE (or TAE) (typically one devoted to this task, can be difficult to clean)

  1. Mix agarose in an Erlenmeyer flask, add the agarose by taco-ing the weigh boat (or weighing paper) and carefully pouring the powder as to not get it stuck to the side of the flask. Pour up to your desired volume with new 1X TBE (or TAE)

  2. Put a scrunched Kimwipe in the mouth of the flask and put in the microwave for 1 minute. Every 20 seconds open microwave and swirl flask. Use glove because glass gets hot and don’t be afraid to check too often because it can boil over

  • tip: Microwaving time can vary, so keep adding time until when you look at the liquid when swirling it is completely clear and there are no “flakes”

  1. Cool on the bench top for 1 – 2 minutes

  2. Add 5 ul ethidium bromide and swirl the flask to mix. Let gel cool up to 5 minutes in the flask before pouring into the gel tray, if you can safely hold it, that’s a good sign it’s ready

  3. Set up gel cast mold, place small tray in the gel box sideways and make sure there is an air-tight seal with the rubber noodles

  4. Pour ethidium bromide solution to the small tray

  5. Put desired comb size into the tray

  6. Using a pipette tip, move any bubbles to the side of the tray

  7. Let harden until opaque and cool ~30min

  8. Take out and orient the gel with the comb side to the top of the box. Take the comb out of the hardened gel

  9. Pour enough “used” TBE (or TAE) buffer into the gel box to cover the gel with a thin layer of liquid and make sure there are no bubbles

  10. In a new strip tube (or Qubit tubes), label each with sample ID and add 5μl aliquots of your DNA samples. Add 1μl of loading dye (i.e. BioLabs Quick loading dye 6X Purple) to each sample and vortex and spin down to mix. You now have a 6ul sample ready for gel electrophoresis

  11. Add 5μl of the appropriate ladder to the first well in the gel (i.e. BioLabs 100 bp Quick Purple ladder)

  12. Add your samples to each well (6μl). Make sure you have made a map in your notebook that shows the order of samples

  13. Make sure gel is set up to “run towards red”. Red = positive. Since DNA is negatively charged, the smaller fragments with run further down the gel toward red than larger molecular-weight DNA fragments.

  14. Plug black cable into black insert and red cable into red insert of the gel box

  15. Turn box on and make sure voltage is set to 100V. Set time for 30-60min.

    Tip: running for longer periods at lower voltage can increase the resolution of bands.

  16. Once done, take the tray out of the box and using a Kimwipe, slide the gel into a kimwipe. Bring over to the transilluminator for imaging

  17. Place the gel in the SynGene Gel Box and start the GenSys software

  18. Wait to connect to the UV reader – click on gel – DNA agarose gel – ethidium bromide – ‘next’. Allow the picture to be capture and adjust for the best resolution. Save as .jpeg and change to 300 dpi.

  19. Remove the gel with gloves and kimwipe and dispose in the ethidium bromide waste (under the bench). Dispose the “used” 1x TBE in the gel plate into the same ethidium bromide waster container.

    Tip: the “used” 1x TBE can be reused several times before compromising gels beofre disposed. If quality is fine, pour the 1x TBE in the gel carefully using a funnel into the 1x TBE earlenmeyer flask.

  20. Email your jpeg (or upload to desired google drive folder) and turn off the SynGene transilluminator software and computer.