Skip to content

Agarose Gel Electrophoresis

Bench manual for routine agarose gel electrophoresis of DNA samples, PCR/qPCR products, and RNA sample checks.

Buffer1X TAE
Routine gel1.5%
Small tray50 mL
Run time25-60 min

Before Starting

Hot agarose

Handle melted agarose carefully. Cool the flask until it feels warm, not hot, before adding stain and pouring the gel.

Stain choice

Use GelGreen for DNA samples and GelRed for RNA samples. Add stain only after the agarose has cooled for 10-15 min.

Routine setup

Use a 1.5% gel for most routine checks. Use a 2% gel when you need better separation of small fragments or PCR/qPCR products.

Materials

Reagents

  • 50X TAE buffer
  • DI water
  • Agarose
  • GelGreen for DNA gels
  • GelRed for RNA gels
  • 6X loading dye
  • 1 kb DNA ladder for genomic DNA or larger DNA fragments
  • 100 bp DNA ladder for PCR/qPCR products

Equipment and Consumables

  • Gel tray and comb
  • Electrophoresis chamber
  • Power supply
  • Microwave-safe flask or bottle
  • Heat-resistant gloves
  • Micropipettes and tips
  • Bio-Rad GelDoc Go system
  • UV/stain-free tray

Buffer and Gel Calculator

Info

Large trays use three times the small-tray recipe.

Use 1 for a small tray, 3 for a large tray.

Setup 1X TAE Agarose for 1.5% Agarose for 2% GelGreen or GelRed
Current gel 50 mL 0.75 g 1.00 g 5 µL
1X TAE batch 50X TAE DI water Final volume
Standard batch 20 mL 980 mL 1 L

Part A: Prepare 1X TAE Buffer

~5 min

  • Label a clean bottle as 1X TAE.
  • Add 20 mL 50X TAE buffer to the bottle.
  • Add 980 mL DI water.
  • Mix well before filling the gel chamber or preparing agarose.

Part B: Make the Agarose Gel

~20-30 min

Small Tray Recipes

Gel percentage 1X TAE Agarose Stain after cooling
1.5% 50 mL 0.75 g 5 µL GelGreen or GelRed
2% 50 mL 1.00 g 5 µL GelGreen or GelRed
  • Choose the gel percentage: 1.5% for most samples or 2% for small fragments.
  • Add the correct amount of agarose and 1X TAE to a microwave-safe flask.
  • Microwave in 30 sec intervals, swirling carefully between intervals, until the agarose is fully melted.
  • Cool for 10-15 min, until the flask feels warm but not hot.
  • Add GelGreen for DNA samples or GelRed for RNA samples. Mix gently without introducing bubbles.

Do not add stain to very hot agarose

Let the agarose cool first. Hot agarose increases burn risk and can reduce stain performance.

Part C: Cast the Gel

~20-30 min, including solidification

  • Place the gel tray in the casting setup.
  • Pour the warm agarose into the tray.
  • Insert the comb to form sample wells.
  • Allow the gel to solidify completely before removing the comb.
  • Place the gel in the electrophoresis chamber and cover it with 1X TAE buffer.

Part D: Load Samples

~10-20 min

DNA Samples

  • Mix DNA sample with 6X loading dye at a 5:1 ratio, such as 5 µL DNA + 1 µL loading dye.
  • Load 5 µL 1 kb DNA ladder in the first well for size reference.
  • Load 5 µL of each prepared DNA sample.

PCR/qPCR Products

  • If the PCR/qPCR product is colorless, mix with loading dye at the same 5:1 ratio.
  • If the PCR/qPCR product already contains loading dye, load it directly.
  • Load 5 µL 100 bp DNA ladder in the first well for size reference.
  • Load 5 µL of each prepared PCR/qPCR product.

Part E: Run and Image the Gel

~25-60 min

Tray Voltage Run time
Small tray 80-85 V 25-30 min
Large tray 90-100 V 45-60 min
  • Connect the chamber to the power supply and confirm the samples will run toward the positive electrode.
  • Run the gel using the voltage and time range for the tray size.
  • Watch the dye front and stop the run before the dye reaches the end of the gel.
  • Image GelGreen DNA gels or GelRed RNA gels on the Bio-Rad GelDoc Go system using the UV/stain-free tray.

Bench Record

Date
Operator
Sample IDs
Sample typeDNA / PCR / qPCR / RNA
Gel percentage1.5%
Tray sizeSmall / Large
Stain usedGelGreen / GelRed
Ladder used1 kb / 100 bp
Loading volume5 µL
Voltage
Run time
GelDoc image file
Notes

Bench Notes

Notes are saved locally in this browser.

Stop Points

Stop and ask before:

  • Loading samples if the gel has not solidified completely.
  • Running the gel if the chamber has too little 1X TAE to cover the gel.
  • Continuing if samples appear to run in the wrong direction.
  • Imaging if the stain used or imaging tray is unclear.

Safety Notes

Gel stain and UV exposure

Wear gloves when handling stained gels. Avoid skin contact with GelGreen- or GelRed-stained gels. Follow GelDoc safety procedures and do not expose skin or eyes to UV light.

Waste

Dispose of GelGreen- or GelRed-stained gels in the red biohazard bag.