Aug 12, 2022
Agarose Gel Electrophoresis (Instructor Protocol)
This protocol is a draft, published without a DOI.
- 1University of Wisconsin - Stout
- Yeast ORFans CURE
Protocol Citation: Brian Teague 2022. Agarose Gel Electrophoresis (Instructor Protocol). protocols.io https://dx.doi.org/
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: August 11, 2022
Last Modified: August 12, 2022
Protocol Integer ID: 68526
Keywords: agarose gel electrophoresi, instructor protocol, gel, protocol, instructor, included several common student error, several common student error, student
Abstract
This is the instructor protocol for
The protocol is pretty straightforward, but I have included several common student errors to watch out for!
Image Attribution
Dr d12, CC BY-SA 3.0 , via Wikimedia Commons
Materials
LAB buffer (recipe linked in the 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.
Wear appropriate personal protective equipment (PPE), including a lab coat, nitrile gloves and safety glasses.
Make the LAB buffer
Make LAB buffer following this recipe:
For a class of 24, I usually prepare 6x 1 L bottles of 1X LAB. We go through a lot -- there are three gels that get run over the course of the semester, plus repeats.
Instructor Tips & Common Student Errors
Instructor Tips
- The benefit of using LAB is the ability to run gels at higher voltage -- our "mini" gel tanks go at 120 V for 20 minutes and get pretty decent gels. It is totally possible to cast, load and run an LAB gel in 2 hours.
- I usually demonstrate the proper assembly of the gel tank
- Gels with Sybr Safe are fine in the refrigerator for 48 hours, but much longer than that and the stain degrades too much. That is -- a gel cast on Tuesday will be fine for a lab on Thursday, but not the other way around. To store a gel, put it in a zipper sandwich baggie and add a splash (not more) of LAB buffer. Label and store at 4 °C in the refrigerator.
- A common student error is to forget the DNA stain. But all is not lost -- you can post-stain these gels. Put the gel in a small container (a tip box lid is often fine), cover with LAB, add 5 µL of Sybr Safe, and put on a slow rocker or orbital shaker.
Common Student Errors
- Forgot to add the DNA stain
- Didn't 100% dissolve the agarose (it should be CLEAR AS WATER, no flecks floating around in it)
- Ran the gel backward (remember, "run to red")
- Only ran 1 µL of the restriction digest (instead of the whole digest)
- Alternately, ran the entire PCR (instead of just 1 µL ) -- leaving not enough to purify.