Gel electrophoresis_50758709

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title: "TheNessLab : Gel electrophoresis"

1. TheNessLab
2. The Ness Lab Wiki
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TheNessLab : Gel electrophoresis

Created by Ahmed Raza Hasan, last modified on Jan 05, 2021

This specific method of electrophoresis was taught to me by grad students in Adriano Senatore's lab. The rigs/power packs in the lab are shared with Adriano, so treat the equipment with care.

Additionally, some electrophoresis etiquette tips from Adriano:

• Please ensure that you empty and rinse rigs after use.
• Some of the gel casting trays are starting to crack. This is caused by pouring hot molten agarose solution into the trays without cooling. Please cool your molten agarose solutions prior to pouring into the trays.
• Please also clean up the area after use, including wiping down the counter and rinsing your flasks containing residual agarose and ethidium bromide.
• Please ensure that you turn off the power packs after use.

Making the gel

Protocol

1. Prep TAE 1x if needed.
   • stock TAE is 50x
   • add 20 mL 50x TAE to 980 mL MilliQ to create 1 L 1x TAE
2. Mix agarose and TAE 1x in an EtBr designated flask to create gel solution. Swirl flask.
   • 1.5% agarose - 2.25 g in 150 mL TAE 1x
   • 3% agarose for longer runs (e.g. when imaging shorter fragments) - 4.5 g in 150 mL TAE 1x
3. Microwave flask for 1 minute and 30 seconds uncovered. Watch flask - as soon as flask starts bubbling over with rising foam, stop microwave (though 1.5 min is usually just enough for 3% gels). If solution is not clear, microwave again for a shorter period of time - once solution is clear, it's ready.
   • Make sure to use thermal gloves to handle flask from here. It's going to be burning hot to the touch.
4.  Let solution cool.
   • Either leave flask at room temp for ~15 minutes, or run side of flask under cold water. I like to periodically (and very carefully) check the heat of the flask against the side of my arm until I can hold it there for several seconds.
   • Careful to avoid letting the solution cool so much it starts solidifying. Pouring too late will make for an uneven gel that will image very poorly, while pouring too early (with the solution still too hot) will cause the gel cast to warp.
5. While the solution is cooling - in gel rig, lift gel cassette and stick it back in rotated 90 degrees. This creates a seal and leaves out the remaining buffer. Make sure the seal is tight. Add gel comb(s) as needed and use pipette tips to seal them in place if needed.
6. Once solution is cool, add ~7-8 uL EtBr directly into the solution. Swirl the flask to mix until EtBr has dissolved.
   • Directly into the solution. Make sure the tip is immersed.
7. Carefully pour gel solution into cassette. Pop/redirect any bubbles with a stray tip. Let dry + solidify for 15-20 minutes.
   • Immediately fill out the flask with water completely and leave in sink. We'll come back to this.
   • I usually use this downtime to refill tips as needed and/or add loading dye to my samples (see below).
8. When the gel has solidified, remove gel comb. The gel will turn increasingly translucent as it dries; when it looks uniformly translucent, it's ready. Careful to grab the sides of the comb and pull directly upwards - do not jiggle it from side to side.  Lift gel cassette + gel back out of the rig and place back in with the correct orientation. The gel should be submerged in buffer - add more 1x TAE if needed.

Materials

• agarose (vol. depends on gel size)
• 1x TAE (vol. depends on gel size)
• EtBr
• flask (designated for EtBr usage)
• p10 pipette
• microwave

Loading and running the gel

Protocol

1. Make sure gel is submerged in 1x TAE buffer.
2. Use parafilm technique to mix DNA ladder with loading dye. Carefully load DNA ladder into wells as needed.
3. If samples aren't already mixed with loading dye, use parafilm technique to mix and load samples on a sample-by-sample basis. Add 8-10 uL sample (vol. includes loading dye) per well.
   • If I'm not going to use the PCR product for anything besides the gel, I usually just add loading dye directly to the PCR product itself. I add 4 uL 6x loading dye added to 20 uL PCR product and mix. This can be sped up quite a bit with multichannel pipettes. This is very useful for quick assays.
4. Add ~5-7 uL EtBr to buffer in gel rig directly below the gel. Swirl left and right with pipette tip to dissolve EtBr.
5. Add ~5-7 uL EtBr to buffer in gel rig directly above the gel. Swirl left and right with pipette tip to dissolve EtBr.
   1. I've been separately told that this addition above the gel doesn't make a huge difference, but I still tend to do it.
6. Connect gel power supply to rig. Make sure electrodes are in the right orientation.
7. Run gel at required voltage. As soon as the power supply is turned on, look at the top end of the gel rig — there should be a 'burst' of evenly spaced bubbles coming out of the top edge, and not the bottom. If not, your elctrodes are reversed.
   • 4V/cm (ie 80-100 V) for 1-1.5 hours is usually a good balance for most purposes.
   • For smaller DNA products, use lower voltages and run for longer. I run 3% gels at 65-70 V for 3 hours to image bands that are ~100 bp in size and have ~15 bp size differences between them.
8. Remember that flask we heated the agarose in and filled up with water? Rinse it out a few times and leave it up to dry.

Materials

• gel in gel rig
• 1x TAE
• EtBr
• p10 pipette
• gel power supply
• PCR'd samples
• 6x loading dye
• DNA ladder
• parafilm

Imaging the gel

Protocol

1. Make sure you've booked the GelDoc on the 3rd floor in advance. You'll need no more than the base 15 minute slot.
2. Stop gel power supply, turn it off, and remove electrodes.
3. Remove gel cassette and gently place gel on styrofoam (or other) tray.
   • I usually put a few napkins on the tray first to absorb all the liquid coming out.
4. Cover gel with napkins and take over to the gel imager (3rd floor core facility)
5. Place the gel inside the imager. Imaging software should be open on the computer already. Select New Protocol → Nucleic Acid → EtBr.
   1. I usually disable the 'highlight saturated pixels' option, which is on by default for some reason.
6. Use the software to help position the gel. The position gel will turn on a standard (non-UV) light, so you can keep the door open and reposition the gel. There are grid lines in the preview to help you line up the gel as well.
7. Close the imager door and scan the gel.
8. Celebrate/panic/grieve/mourn after looking at the results.
9. Save image to the lab folder - make sure to use an informative file name that has the date at minimum. Print to the Mitsubishi printer if desired.
10. Take the gel back out. Make sure to clean the inside of the imager.
11. Bring the gel and tray back downstairs. Dispose of the gel, your gloves, and napkins in a specially marked EtBr waste bucket.

Document generated by Confluence on May 22, 2024 11:44

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