TITLE: 2% Agarose Gel
AUTHORS: George  Testo

[DESCRIPTION]
Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar. The proteins may be separated by charge and/or size (isoelectric focusing agarose electrophoresis is essentially size independent), and the DNA and RNA fragments by length. Biomolecules are separated by applying an electric field to move the charged molecules through an agarose matrix, and the biomolecules are separated by size in the agarose gel matrix.

An Agarose gel is easy to cast, has relatively fewer charged groups, and is particularly suitable for separating DNA of size range most often encountered in laboratories, which accounts for the popularity of its use. The separated DNA may be viewed with stain, most commonly under UV light, and the DNA fragments can be extracted from the gel with relative ease. Most agarose gels used are between 0.7–2% dissolved in a suitable electrophoresis buffer.

[BEFORE_START]
You may store the SYBR™ Safe DNA gel stain at any temperature between 2°C to 25°C. SYBR™ Safe in DMSO freezes at low temperatures; therefore, the product must be completely thawed and mixed before using. Repeated freeze-thawing has minimal impact on product performance.

[STEPS]
SECTION: Pouring a Standard 2% Agarose Gel
1. Measure 1g of Agarose.
SECTION: Pouring a Standard 2% Agarose Gel
2. Mix agarose powder with 49mL of DI water and 1mL of 50x TAE in a microwavable flask.
SECTION: Pouring a Standard 2% Agarose Gel
3. Microwave for 1-3 minutes until the agarose is completely dissolved.

 


 

SECTION: Pouring a Standard 2% Agarose Gel
4. Let agarose solution cool down to about 50°C for about 5 min. 

SECTION: Pouring a Standard 2% Agarose Gel
5. OPTIONAL: Add 5uL of SYBR Safe to the flask.
 

 



SECTION: Pouring a Standard 2% Agarose Gel
6. Pour the agarose into a gel tray with the well comb in place.
SECTION: Pouring a Standard 2% Agarose Gel
7. Place newly poured gel at 4 °C for 15 min or let sit at room temperature for 30 minutes, until it has completely solidified.
 


SECTION: Loading Samples and Running an Agarose Gel
8. Add loading buffer to each of your ladders and DNA samples.
SECTION: Loading Samples and Running an Agarose Gel
8.1. Make your Ladder Dilution: 

Make your Ladder Dilution: Molecular Grade Water + 10uL of 1kb Ladder
SECTION: Loading Samples and Running an Agarose Gel
9. Once solidified, place the agarose gel into the gel box (electrophoresis unit).
SECTION: Loading Samples and Running an Agarose Gel
8.2. On a small sheet of parafilm, pipette mix at least 10 µL of ladder or sample with 2 µL of 6x Loading Dye for a total volume of 12 µL for gel loading. For lower concentrations of DNA, pipette mix at least 15 µL of ladder or sample with 2.5 µL of 6x Loading Dye.

SECTION: Loading Samples and Running an Agarose Gel
10. Fill gel box with 1x TAE (or TBE) until the gel is covered.
SECTION: Loading Samples and Running an Agarose Gel
11. Carefully load a molecular weight ladder into the first lane of the gel.
SECTION: Loading Samples and Running an Agarose Gel
12. Carefully load your samples into the additional wells of the gel.
SECTION: Loading Samples and Running an Agarose Gel
13. Run the gel at 80-150V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
 

SECTION: Analyzing Your Gel
14. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
 

SECTION: Analyzing Your Gel
15. Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as “bands” due to their appearance on the gel.