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DNA Electrophoresis

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to Create a 2% Agarose Gel and Cast it to perform DNA Electrophoresis.

Reagents and Materials

Things to Note before starting

Estimated Timing

Casting a gel can take ~20 minutes for the Gel to solidify. Running the gel takes ~20-40 minutes depending on the separation you are aiming for.

Procedure

Casting the Gel

  1. Uncap the RM034 - List of Commonly Used Lab Buffers#2% Gel Agarose Solution|2% Gel Agarose Solution and Microwave for 1 minute per 100mL to melt the solidified gel. Gently Swirl the solution every minute. Use hot mitten's to touch the glass to avoid burns.
  2. Place a Gel Loading container perpendicular to the flow of current of a Gel Electrophoresis box so that the ends of the loading container are sealed by the walls of the Electrophoresis box.
  3. Place the gel comb into its indicated groove.
  4. Carefully Swirl the RM034 - List of Commonly Used Lab Buffers#2% Gel Agarose Solution|2% Gel Agarose Solution to ensure it has completely melted. If the solution appears cloudy, microwave for longer until the solution appears translucent.
  5. Carefully pour the RM034 - List of Commonly Used Lab Buffers#2% Gel Agarose Solution|2% Gel Agarose Solution into Gel Loading Container. Ensure that the solution covers the bottom of the of the container and is tall enough to go halfway up the teeth of gel comb.
  6. Let the gel sit for ~20 minutes until it solidifies.

Preparing Samples for Electrophoresis

  1. Label PCR tubes with numbers corresponding to each sample
  2. Add 5uL of PCR Product to each PCR Tube
  3. Add 5uL of 6X DNA Loading Dye to each tube 1
  4. Spin down PCR tubes in a tabletop PCR Tube centrifuge

Running the Electrophoresis

  1. Check to see if the Gel has solidified by lightly touching it. The color of the gel should be a cloudy opaque white when it solidifies.
  2. Carefully remove the Gel Comb from the solidified gel.
  3. Slowly shimmy the Gel Loading Container out of the Electrophoresis box.
  4. Rotate the Gel Loading Container so that it is parallel to the flow of current. Insert the Loading Container by lubricating the rubber strip against the walls of the Electrophoresis Box with TAE Buffer.
  5. Add 1X TAE Buffer to the Electrophoresis Box until the buffer level is above the gel.
  6. Add 10uL of Sample into its corresponding well on the gel. Be sure to place the pipette inside the well before dispensing, and make sure to not puncture the bottom of the gel. 2
  7. Add 5uL of DNA Ladder to the wells on either end of the Gel.
  8. Apply the lid to the Electrophoresis box in such a way that the samples are closest to the Black Wire and the bottom of the Gel is closest to the Red Wire. 3
  9. Connect the Wires to the Power Controllers in the Shelf above the Gel Bench
  10. Turn on the power, set the voltage to 120V
  11. Ensure that current is running by looking for small bubbles being formed in the reservoir of the Electrophoresis Box.
  12. Run on voltage until the loading dye shows the samples approaching the bottom of the gel (~30 minutes)

Next Steps

A photo of the gel can be taken by the machine in the dark room across the hall from the Cold Room. This photo should be saved for your records.

Appendix

Recipe's for Buffers

2% Gel Agarose Solution

This reagent is used to cast DNA Electrophoresis Gels. It should be stored at room temperature on the shelves above the Gel Bench. The solution should be microwaved to allow the solution to melt before being used.

  • 3.6mg of UltraPure Agarose Powder
  • 200mL of 1X TAE Buffer
  • 100mL of GelRed Nucleic Acid Stain
  • Solution should be mixed by swirling and then microwaved for 3 minutes. The Solution should be periodically swirled every minute to help the agarose dissolve. The solution should appear translucent when the agarose has completely dissolved.

Footnotes

  1. The Amount of Gel Loading Dye ranges from 1-5uL per 5uL of DNA. The amount you add corresponds to how easy it is to see the traveling of the current as this dye corresponds to the leading edge of traveling DNA. ↩

  2. This can take a bit to get used to. There is no magic trick to placing the pipette in just right. Feel around with the pipette to get adjusted to the dimensions of the well. With practice, you will become more comfortable loading the wells. ↩

  3. Remember the familiar pneumonic "DNA Runs to Red". Therefore, the DNA should want to move towards the Red Wire Terminal. ↩