Experimental Protocol for DNA Agarose Gel Electrophoresis

by K Chico, Jessica Piczon

Gel electrophoresis is the fractionation of a protein, DNA, or RNA based on the size, shape, and/or net charges of the macromolecules. Briefly, charged particles are forced to migrate from one direction to another under the influence of an externally applied electric field. The gel matrix acts as a molecular sieve, and slows movements by inducing interactions within molecular particles. The opposing interactions of the electrical force (drawing the molecules in one direction) and sieving (pulling particles down into the matrix) result in differential migration rates for constituent proteins of a sample.

Fig. 1

Comparison of DNA detection in 1% agarose gel in TBE buffer using Gelite™ Safe, SYBR® Safe, and EtBr. Two-fold serial dilutions of 1 kb DNA ladder were loaded in amounts of 86 ng, 43 ng, 21.5 ng, 10.7 ng, 5.3 ng, 2.6 ng, 1.3 ng, and 0.5 ng from left to right. Gels were imaged using a 300 nm transilluminator in ChemiDoc™ Imaging System (Bio-Rad®).

Gel electrophoresis is used diversely across many fields of science, including forensics, molecular biology, genetics, biotechnology, and pharmacology. Among many other things, gel electrophoresis can be used to analyze PCR products, determine total protein content. quantitate proteins within a sample, assess post-translational modifications, analyze genes associated with specific illnesses, and assess the efficacy of antibiotics, terpenoids and steroids.

Key Parameters

Gel electrophoresis box setup
Transilluminator

Reagents

Agarose tablet or powder
0.5X TBE or TAE buffer (depending on projected size of DNA fragments)
Ultra pure or double distilled water
Nucleic acid stain (Ethidium Bromide (EtBr), SYBR Green, Gel Red, Gelite Safe, etc.)
Loading dye
Ladder
DNA Samples

Sample Experimental Protocol (for DNA Gel Electrophoresis using Agarose Gel)

Agarose Gel Preparation and Setup

Prepare TBE buffer by adding 20 mL of 0.5X TBE Buffer to 980 mL water.
- To make a 1% gel, combine 0.5 g agarose and 50 mL of 0.5X TBE Buffer in a beaker. Swirl the beaker to mix.
While waiting for the agarose to dissolve, set up the gel box for casing the gel.
1. Open the gel box and ensure the dams on each side of the box are installed correctly.
2. Assemble the gel tray into the cassette and install the comb.
  Note: Ensure the comb is in the correct orientation, with the negative/black electrode above the wells so that the DNA runs toward the positive/red electrode.
Head back to your agarose, and make sure tablets are fully dissolved.
1. Microwave solution for bursts of 20-30 seconds. Remove the beaker with gloves (it will be warm) and swirl. Microwave for longer if there is undissolved agarose.
2. Allow the solution to cool for 1 min.
  Note: Place a folded paper towel on top of the beaker when microwaving to prevent steam from escaping.
  Note: Avoid heating the gel for too long. Heat in short bursts, stop when bubbles appear, and swirl the solution. If the solution is overboiled, the gel will have a higher percentage of agarose with a high ionic concentration.
Add your fluorescent stain.
- Add either 1 μL of EtBr or 3 μL of a similar nucleic acid stain to the melted agarose. (amount varies depending on detection limits and final concentrations) Mix well by swirling the beaker until the stain is uniformly dispersed.
  Note: EtBr is a carcinogen and must be handled with care.
Once the gel is around 50 ℃, pour the gel slowly into the tray.
Note: Avoid pouring gel >70°C. This may lead to the gel box and comb warping.
Note: Avoid pouring too fast, as this might create bubbles within the gel. Bubbles can be carefully popped using a small pipette tip.
Let the gel sit at room temp for 30 mins to solidify.
- Alternatively, the gel tray may be placed in the fridge to speed up the process.
  Note: Ensure the gel box is on a level surface to ensure thickness consistency.
Once the gel has solidified, carefully remove the comb and the buffer dams.
Note: When removing the comb, make sure to avoid piercing the gel.
Once solid, pour 0.5x TBE buffer over the gel until the buffer covers 2-3 mm above the gel.

Loading the Dyes and Gel

Add 5 μL of loading dyes to each sample.
Note: Loading dyes add weight to the sample and help it sink in the well.
Note: If your master mix already contains a loading dye, this step may be skipped.
Load each sample into their respective wells.
1. Load an appropriate ladder into the first lane well. Generally, 5 μL can be used.
2. For most applications, load 20-100 ng of DNA into each other lane well.
  Note: An appropriate ladder should be chosen, based on the weight of your samples and expectation for fragments.
  Note: While loading, avoid bubbles in the samples.
  Note: To better spot wells, place the gel tray onto a darker surface to increase contrast.
  Note: Though usually wells of the gel are loaded left to right, load your gel based on what hand you work with. Right handers work left to right, and left handers work right to left. This will help avoid contamination.
  Note: Prior to/while loading, make sure to note which sample is in each well.

Fig. 2

An illustration of gel electrophoresis workflow (figure made in BioRender).

Running the Gels

Place the lid on the cassette and ensure the red and black wires are connected to the matching red and black electrodes on the cassette.
1. Gel can be run at a variety of time and voltage settings depending on the size of samples and desired separation.
2. For most samples, 50V for 30-50 min will work.
Once the run is complete, disconnect the gel box.
Open the lid and discard the buffer.
The tray and gel can now be imaged with transillumination/UV.

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