Agarose gel electrophoresis of dsDNA

Goal:

To separate dsDNA fragments of different molecular weight for analysis or subsequent use in cloning

Introduction

Agarose gels help you visualize DNA. Cool!
You can make agarose from 0.5% to even 3%, by mass. 0.7% shows separation of large fragments (5-10kb) and 2% shows good separation of small fragments (0.2k-1k). Keep in mind the gelliness/solidness positively correlates with more agarose; 0.5% gel will be floppy and fragile.

Agarose % guide

Agarose (g/100mL) DNA resolution (kb)
0.5 1-30
0.7 0.8-12
1.0 0.5-10
1.2 0.4-7
1.5 0.2-3

Taken from Agarose gel electrophoresis.

Materials

  • TAE or TBE, 1x – 50 mL is good for a little gel but you have to determine empirically
    • Don’t run a TAE gel in TBE
    • Add extra volume of TAE to compensate for boiling
    • If gel purifying: add 1 mmol/L guanosine to the TAE/TBE. This does not affect ligation and other downstream steps, however it will protect sticky ends. (See references)
  • 0.5-2% agarose, by mass (e.g. for 1%, dissolve 0.5g agarose in 50 mL)
  • Ethidium Bromide (for 10mg/mL, use 1uL per 50mL TAE)

Pouring, Loading, and Running your TAE gel

  1. Measure desired mass of agarose for the particular percentage gel you would like to make:
    • 0.9% = 0.45g in 50mL
    • 1.2% = 0.60g in 50mL
  2. Transfer the agarose to a 125 or 250mL erlynmeyer flask
  3. Add 50mL 1x TAE. Swirl vigorously. It will be cloudy
  4. Microwave until it boils (~1 minute). Take it out and swirl (CAUTION: it’s hot). Keep microwaving until the agarose flakes are minimized.
  5. Let it cool on your bench until the flask is just warm to the touch (~10-15 minutes).
  6. While cooling, set up the gel pour-er with the ladder already in place.
  7. Add 2.5uL of Sybr Safe. Swirl and pour immediately.
    • Minimize bubbles by pouring gently. If there are bubbles, move them to the side with a pipette tip.
    • This will give you a “0.5x” dilution of the Sybr Safe. In our experience this has been plenty to visualize the gel. If you’d like to try a 1x dilution, add 5uL.
    • If you are using a volume of TAE different from 50mL make sure to adjust the volume of CyberGreenI accordingly.
    • CAUTION: Sybr Safe is “not” a mutagen, but since it still interchelates DNA, use with caution.
      • Only use this chemical in the gel loading area.
      • Minimize breathing in vapors by waiting for your gel solution to cool before adding CyberGreen.
  8. Let the gel solidify (~10-20 minutes). It is always better to wait longer!
  9. Load samples.
    • Putting a black block under the loading area can help you see the lanes.
    • If you have less samples than there are lanes, I recommend loading them in the center of the gel. We’ve gotten much better resolution in the center lanes.
  10. Put the top of the electrophoresis box on and run your gel at 100V for ~30 minutes.

How fast will my gel run?

Generally 30 minutes at 100V is a good way to start. If there are 2 bands you would like to resolve, you can always plop the gel back in for another couple of minutes.

Higher voltage and your DNA will migrate faster. It will also heat up the buffer and gel, and bands may appear diffuse.

Imaging your gel

Our gel doc is located in 5204.

  1. Turn on the camera
  2. Open the program for the GelDoc
  3. …
  4. Put your gel in the gel doc
  5. Use “live imaging” with epi-white light (filter set = none) so you can see where your gel is and focus the camera properly.
  6. Close the door to the gel doc and click the “uv” button. Change the filter set to CyberGreen
  7. Click (…)
  8. Use the auto expose function
  9. Save as a jpeg

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