PREP: Electrophoresis Buffer & Agarose Gel
Name:__________________________________________Date:_______________
Intro:
Agarose gels are typically used to separate and analyze DNA molecules ranging in length from
about 500 to 25,000 base pairs (bp). The ability of a gel to separate molecules is called “resolving
power” and is mainly determined by the concentration of agarose in the gel. Most gels have
concentrations between 0.6% and 3.0% agarose in buffer.
Agarose gels are prepared by dissolving powdered agarose in a certain volume of electrophoresis
buffer. The agarose-buffer mixture has to be boiled for the agarose to go into solution.
Electrophoresis = process using electric field to move negatively charged DNA to migrate to
positive electrode.
The concentration depends on the type of molecules to be analyzed. The longer the molecules in a
sample, the less concentrated the gel should be. As the concentration increases, the agarose threads
are pushed closer together, making it difficult for larger molecules to move through them.
For Princeton Gel Boxes: 30 ml gel bed & 150 ml of 1x buffer for chamber; 180 mL buffer
TOTAL (Make buffer total/group 200 ml for wiggle room).
Procedure:
1) Prepare Electrophoresis Buffer for Entire Class:
*This is usually just a dilution: ( V1C1 = V2C2)
V1 = volume needed of buffer concentrate
C1 = concentration of stock concentrate
V2 = volume needed for experiment
C2 = diluted concentration
Example: If stock buffer is 50x and you need 500 ml of 1x, determine volume of stock concentrate
needed? ________________ Show work:
V1
X
C1
50x
V2
500 ml
C2
1x
Recipe: ____________ml of 50x Buffer and _________mL distilled H2O
*Now that you’ve prepared enough buffer for the class, you are ready to make the gel.
2) Prepare Agarose Gel
X g of agarose powder = % Gel
Total volume (mL)
* Agarose is a purified form of agar, a complex polysaccharide derived from seaweed.
Molten 1.0% agarose is kept in a 65oC water bath so it will not solidify!
*Example: Prepare a 1% gel with a volume of 30 ml using 1x electrophoresis buffer:
_________g agarose buffer + 30 ml buffer (for convenience)
Princeton Satellite Site
Spring 2011
K. McKone
PREP: Electrophoresis Buffer & Agarose Gel
A. Preparing Gel:
________1) Obtain clean 50 mL Erlenmeyer flask (or whatever is available).
________2) Weigh out _______g agarose powder in a weigh boat and add to flask.
________3) Add required buffer slowly to agarose, swirling to mix. _________mL buffer
________4) Dissolve by heating on a hot plate until solution is clear.
________5) Solution must cool to 65oC before pouring (placing your hand under bottom of flask w/out
discomfort is an indicator).
*Do NOT allow it to cool below 60 oC or it will start to solidify too soon.
________6) Prepare gel chamber by following directions below:
B.
Pouring the Gel:
1. _________Set up the gel box by placing a stopper in each of the slots at the end of
the gel bed. Be sure that the side of the stopper next to the gel box forms a right
angle with the gel bed.
2. _________Seal the ends of the gel bed before pouring the agarose. Use the plastic
pipette to lay down a narrow bead of melted agarose along the joint between the
black stopper and the gel bed along both ends.
Seal with Agarose
Gel Bed
3. __________Put an 8-tooth comb into the end of the gel that is nearest the black
(negative) electrode. The red stripe under the comb will allow you to visualize
the wells after the comb has been removed.
4. __________Wait one minute while the bead solidifies, then pour enough agarose
into the gel bed until the level of agarose reaches the top of the bed but does not
overflow (about 30mL). The gel will appear thicker than it actually is – observe
the level carefully and be sure to pour in enough agarose.
5. _________Allow the gel to solidify at room temperature for 10-20 minutes.
Either:
a. Proceed with loading the gels, or
b. Remove stoppers and place gel in plastic bag with a small amount of
buffer. Refrigerate overnight.
6. _________ After pouring, CLEAN FLASK IMMEDIATELY!!!
Princeton Satellite Site
Spring 2011
K. McKone