Jul 11, 2022
Agarose gel electrophoresis
This protocol is a draft, published without a DOI.
- 1University of Wisconsin - Stout
Protocol Citation: Brian Teague 2022. Agarose gel electrophoresis. protocols.io https://protocols.io/view/agarose-gel-electrophoresis-ccs9swh6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in an introductory molecular cell biology laboratory and it’s working for us.
Created: July 06, 2022
Last Modified: July 11, 2022
Protocol Integer ID: 66113
Keywords: dna, agarose, gel, electrophoresis
Abstract
We use agarose gel electrophoresis to analyze DNA samples -- is there DNA present? What size is it?
Image Attribution
Wikimedia user TransControl, https://commons.wikimedia.org/wiki/File:Agarosegelphoto.jpg
Guidelines
This protocol has been optimized so it can be run in a 2-hour laboratory section.
Materials
1X LAB buffer
Note
Make LAB buffer using this protocol:
Protocol
NAME
LAB Agarose Gel Electrophoresis Buffer Recipe
CREATED BY
Brian Teague
AgaroseFisher ScientificCatalog #BP1356 (any biotechnology-grade agarose is fine)
SYBR SafeInvitrogen - Thermo FisherCatalog #S33102 (or another DNA stain)
Quick-Load Purple 1 kb Plus DNA Ladder - 250 gel lanesNew England BiolabsCatalog #N0550S (or another DNA molecular weight standard, pre-mixed with loading dye)
Gel Loading Dye Purple (6X) - 4.0 mlNew England BiolabsCatalog #B7024S
Parafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
Protocol materials
Gel Loading Dye Purple (6X) - 4.0 mlNew England BiolabsCatalog #B7024S
Materials, Step 13
Parafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410
Materials, Step 12
AgaroseFisher ScientificCatalog #BP1356
Materials, Step 2
SYBR SafeInvitrogen - Thermo FisherCatalog #S33102
Materials, Step 2
Quick-Load Purple 1 kb Plus DNA Ladder - 250 gel lanesNew England BiolabsCatalog #N0550S
In Materials and 2 steps
Safety warnings
Lithium acetate: May cause eye and skin irritation. May cause respiratory and digestive tract irritation. The toxicological properties of this material have not been fully investigated.
Boric acid: May damage fertility. May damage the unborn child.
SYBR Safe DNA stain: Anything that binds DNA is a potential mutagen / carcinogen.
Wear appropriate personal protective equipment (PPE), including a lab coat, nitrile gloves and safety glasses.
Before start
Before starting, make sure you have the DNA samples you'll be analyzing (either PCR or restriction digest) thawed on ice.
Cast the gel
Cast the gel
30m
30m
Using a balance, weigh out 0.5 g agarose in a weigh boat.
In a 250 Erlenmeyer flask, mix 50 mL LAB buffer , 0.5 g AgaroseFisher ScientificCatalog #BP1356 , and 2 µL SYBR SafeInvitrogen - Thermo FisherCatalog #S33102 DNA stain. Swirl to mix.
Note
Use a graduated cylinder to measure out the LAB buffer.
Microwave on HIGH for 00:00:30 ; the solution should begin to boil. Remove from the microwave and swirl.
Safety information
Be careful, it will be very hot! Use a folded-over paper towel and grab the flask at the top to avoid burning yourself.
30s
Look carefully at the contents of the flask while you swirl it. If there are any flecks of undissolved agarose remaining, microwave again for 00:00:15 . Repeat until the solution is completely clear.
Note
It should look as clear as water when you swirl it.
15s
Set the agarose solution on the bench to cool slightly. While it is cooling, insert the black wedges and the gel comb into the gel box.
Note
Make sure the combs are on the side of the gel box near the black electrode.
When the agarose solution is cool enough that you can hold your hand against it for 10 seconds, carefully pour the solution into the gel tray.
Note
It is particularly important that the agarose solution be cool enough, because if it isn't the gel box can be damaged.
Note
If you would like to cool it faster, you can swirl the flask under running cold tap water. Don't let it solidify in the flask!
Wait 00:15:00 for your gel to solidify.
Note
The gel should be solid and uniformly translucent.
15m
Carefully remove the gel combs and the black wedges.
Note
PAUSE POINT -- You can put your gel in a sandwich baggie with a splash of buffer and store it in the refrigerator for up to 48 hours. Longer than that and the DNA stain will degrade.
Run the gel
Run the gel
30m
30m
If you removed your gel from the gel box (or cast it outside of the gel box), put your gel in the box with the wells toward the black electrode.
Pour just enough 1X LAB buffer into the box to entirely submerge the gel.
Below, select whether you're analyzing a PCR or a restriction digest:
To analyze a PCR5 steps
Follow these steps to analyze a PCR on the gel.
Cut a 1 cm x 4 cm (half a square) of Parafilm™ M Laboratory Wrapping Film, 4 in. W x 125 ft. L; (10cm x 38m)Thermo FisherCatalog #1337410 off of the roll.
For each sample (not including the molecular weight standard), carefully deposit 1 µL of Gel Loading Dye Purple (6X) - 4.0 mlNew England BiolabsCatalog #B7024S onto the square of parafilm, then pipette 2 µL of the DNA sample onto the spot. Pipette up and down to mix.
Note
Keep careful track of which spot is which sample!
Carefully pipette 5 µL of Quick-Load Purple 1 kb Plus DNA Ladder - 250 gel lanesNew England BiolabsCatalog #N0550S into the left-most well. Then, pipette your samples mixed with loading dye into successive wells.
Assemble the top of the gel box onto the bottom. Plug the box into a power supply and run the gel at 10for 00:20:00 .
Note
After you start it, look closely at the electrodes in the gel box to make sure there are bubbles. If there aren't, the gel box may be assembled incorrectly or damaged.
Note
The LAB buffer allows us to run the gel at a higher voltage, and for a shorter time, than we would with a traditional buffer like TAE or TBE. However, this decreases the resolution somewhat. For a "prettier" gel, you can run for 30 minutes at 100 volts.
20m
After you have run your gel, image it with a transilluminator.