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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for agarose gel electrophoresis. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/gel-electrophoresis/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Pouring a Standard 1% Agarose Gel]
Measure  of agarose.
0.6 g
*Pro-Tip*Agarose gels are commonly used in concentrations of 0.7%-2% depending on the size of bands needed to be separated -see FAQ section of this protocol. Simply adjust the mass of agarose in a given volume to make gels of other agarose concentrations (e.g.,   of agarose in   of TAE will make a 2% gel).
?. [Pouring a Standard 1% Agarose Gel]
Mix agarose powder with  1xTAE in a microwavable flask. See TAE Recipe.
60 mL
*Pro-Tip*TBE can be used instead of TAE, labs usually use one or the other, but there is very little difference between the two.
Note, make sure to use the same buffer as the one in the gel box (do not mix different buffers and do not use water).
?. [Pouring a Standard 1% Agarose Gel]
Microwave for  to  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.)
CAUTION: HOT! Be careful stirring, eruptive boiling can occur.
*Pro-Tip*It is a good idea to microwave for  - , stop and swirl, and then continue towards a boil. Keep an eye on it the solution has a tendancy to boil over. Placing saran wrap over the top of the flask can help with this, but is not necessary if you pay close attention.
?. [Pouring a Standard 1% Agarose Gel]
Let agarose solution cool down to about  (about when you can comfortably keep your hand on the flask), about .
50 °C
?. [Pouring a Standard 1% Agarose Gel]
Add Midori Green (25,000x) to a final concentration of 1x (about  - of lab stock solution per  gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
2 µl
3 µl
60 mL
?. [Pouring a Standard 1% Agarose Gel]
Pour the agarose into a gel tray with the well comb in place.
*Pro-Tip*Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.
?. [Pouring a Standard 1% Agarose Gel]
Place newly poured gel at  for -  OR let sit at room temperature for -, until it has completely solidified.
4 °C
*Pro-Tip*If you are in a hurry, the gel will set more quickly if you place the gel tray at   earlier so that it is already cold when the gel is poured into it.
?. [Loading Samples and Running an Agarose Gel]
Add loading buffer (6x Purple Loading Dye from NEB) to each of your DNA samples.
Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it settle to the bottom of the gel well, instead of diffusing in the buffer.
?. [Loading Samples and Running an Agarose Gel]
Once solidified, place the agarose gel into the gel box (electrophoresis unit).
?. [Loading Samples and Running an Agarose Gel]
Fill gel box with 1xTAE (or TBE) until the gel is covered.
?. [Loading Samples and Running an Agarose Gel]
Carefully load a molecular weight ladder into the first lane of the gel.
Note, when loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the sample is unloaded, push the pipettor to the second stop and carefully raise the pipette straight out of the buffer.
?. [Loading Samples and Running an Agarose Gel]
Carefully load your samples into the additional wells of the gel.
?. [Loading Samples and Running an Agarose Gel]
Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about -, depending on the gel concentration and voltage.
Note: Black is negative, red is positive. The DNA is negatively charged and will run towards the positive electrode. Always Run to Red.
?. [Loading Samples and Running an Agarose Gel]
Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
?. [Loading Samples and Running an Agarose Gel]
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.
CAUTION: When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat.
*Pro-Tip*If you will be purifying the DNA for later use, use long-wavelength UV and expose for as little time as possible to minimize damage to the DNA.