Sep 27, 2019
Version 2
Agarose gel electrophoresis V.2
- 1University of Cambridge
Protocol Citation: Cristian Riccio 2019. Agarose gel electrophoresis. protocols.io https://dx.doi.org/10.17504/protocols.io.7q7hmzn
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 and it's working
Created: September 27, 2019
Last Modified: September 27, 2019
Protocol Integer ID: 28159
Keywords: agarose, agarose gel, agarose gel electrophoresis, electrophoresis
Abstract
Agarose gel for DNA electrophoresis
This protocol is useful to check the approximate size of a mixture of DNA fragments.
For example, after a HMW gDNA extraction, it is good to check that the DNA we extracted is actually high-molecular weight. Another use is for separating PCR amplicons when checking for a mutation. Primers can be designed in such a way that PCR will yield two fragments of different sizes depending on whether a sequence is present or not. If a deletion is present, then the amplified fragment will be smaller than for the DNA where there is no deletion.
Materials
MATERIALS
Gel Loading Dye Orange (6X) - 4.0 mlNew England BiolabsCatalog #B7022S
TAE Buffer (Tris-acetate-EDTA)Catalog #B49
AgaroseMerck MilliporeSigma (Sigma-Aldrich)
Image Lab SoftwareBio-Rad Laboratories
Autoclave Indicator TapesBio Basic Inc.Catalog #AT011.SIZE.1ROLLS
Ethidium bromide 10 mg/mlMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1510
PowerPac™ HC Power SupplyBio-Rad LaboratoriesCatalog #1645052
agarose gel container
Bio-Rad electrophoresis machine
Bio-Rad Gel Doc XR with Image Lab Software
Microwave oen
HyperLadder 100 bpBiolineCatalog #BIO-33030
HyperLadder 1 kbBiolineCatalog #BIO-33026
Safety warnings
Ethidium bromide is a carcinogen and you should be trained on how to handle it before use. EtBr also needs to be diposed of in a special way.
Get a 250 ml flask and a 250 ml measuring cylinder.
part1.mp4 Add 1 gram agarose to the flask. Add 130 ml TAE in measuring cylinder. Taper container. Put comb in container.
Microwave flask with agarose + TAE for 1 minute. After one minute, check that the solution is homogeneous. If not, microwave in steps of 15 seconds until it is homogeneous.
Move taped and combed container to the cold room along with the quasi-boiling agarose solution. Add 1 microlitre ethidium bromide to the agarose solution. Pour the agarose solution into the container. Make sure there is no spillage. If there is spillage, stop pouring, clean the mess and start again.
Add a small amount of water to the flask, rinse it and dispose of the contaminated water in the liquid EtBr waste through the white material.
Wash flask with water and leave it in the dirty dishes trolley.
Leave the gel to solidify in the cold room. If you want to use the gel the following day, wrap it in a plastic sheet to protect it from desiccation.
2h
Remove tape, put gel in electrophoresis tank. Add 5 microlitres ladder.
Add 2 microlitres loading buffer to 5 microlitres of DNA sample (~ 100 nanograms) and load into gel.
Attach the cables from the tank to the Bio-Rad power machine. Set the programme to 150 V, 400 mA and 30 minutes. Start run.
30m
Check gel every 10 minutes. Put gel in Imager. Open Image Lab. New Protocol, Position gel, zoom in/out. Start protocol. Image is ready on computer. Invert contrast, change contrast, print image. Don't touch computer without gloves. It is contaminated.
Bio-Rad 2019-05-26 15hr 05min.pdf
Bio-Rad 2019-05-26 15hr 05min.pdf Acknowledgments
Acknowledgments
Jérémie Le Pen and other colleagues in the Miska lab
Ulrika Yuan for recording