TITLE: Fast Agarose Gel Electrophoresis-Chem 384/584 
AUTHORS: Ken   Christensen, Addgene  The Nonprofit Plasmid Repository

[DESCRIPTION]
This protocol is for agarose gel electrophoresis. To see the full abstract and additional resources, visit the Addgene protocol page.

[GUIDELINES]
Analyzing your gel
Using the DNA ladder in the first lane as a guide (the manufacturer's instruction will tell you the size of each band), you can infer the size of the DNA in your sample lanes. For more details on doing diagnostic digests and how to interpret them please see the Diagnostic Digest page.

Purifying DNA from your gel
If you are conducting certain procedures, such as molecular cloning, you will need to purify the DNA away from the agarose gel. For instructions on how to do this, visit the Gel Purification page.

Tips and FAQ

How do you get better resolution of bands?
A few simple ways to increase the resolution (crispness) of your DNA bands include: a) running the gel at a lower voltage for a longer period of time; b) using a wider/thinner gel comb; or c) loading less DNA into the well. Another method for visualizing very short DNA fragments is polyacrylamide gel electrophoresis (PAGE), which is typically used to separate 5 - 500 bp fragments.

How do you get better separation of bands?
If you have similarly sized bands that are running too close together, you can adjust the agarose percentage of the gel to get better separation. A higher percentage agarose gel will help resolve smaller bands from each other, and a lower percentage gel will help separate larger bands.

10% Rule:
For each sample you want to load on a gel, make 10% more volume than needed because several microliters can be lost in pipetting. For example, if you want to load 1.0 μg in 10 μL, make 1.1 μg in 11 μL.

[STEPS]
SECTION: Pouring a Standard 1% Agarose Gel
1. Measure .6 g of agarose.
 

SECTION: Pouring a Standard 1% Agarose Gel
2. Mix agarose powder with 60 mL 1xTAE in a microwavable flask. See TAE Recipe.  
 


 

SECTION: Pouring a Standard 1% Agarose Gel
3. Microwave for 1 min to 3 min until the agarose is completely dissolved (but do not overboil the solution, as some of the buffer will evaporate and thus alter the final percentage of agarose in the gel. Many people prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.)
 

 

SECTION: Pouring a Standard 1% Agarose Gel
4. Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 min.
SECTION: Pouring a Standard 1% Agarose Gel
5. Add Midori Green (25,000x) to a final concentration of 1x (about 2 µL -3 µL of lab stock solution per 60 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.





SECTION: Pouring a Standard 1% Agarose Gel
6. Pour the agarose into a gel tray with the well comb in place.
 

SECTION: Pouring a Standard 1% Agarose Gel
7. Place newly poured gel at 4 °C for 10 min-15 min  OR let sit at room temperature for 20 min-30 min, until it has completely solidified.
 

SECTION: Loading Samples and Running an Agarose Gel
8. Add loading buffer (e.g., to6x Purple Loading Dye from NEB) to each of your DNA samples.

  
 


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
10. Fill the gel box with 0.25xTAE (or TBE) until the gel is covered.


SECTION: Loading Samples and Running an Agarose Gel
11. Carefully load a molecular weight ladder into one 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 250-300 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 15 min-25 min, depending on the gel concentration and voltage.
 

SECTION: Loading Samples and Running an Agarose Gel
14. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
SECTION: Loading Samples and Running an Agarose 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.