1. Using Gel Electrophoresis to Study Molecules

2. What is Gel Electrophoresis?
A process that uses electricity to separate charged molecules:
DNA fragments (double stranded)
RNA (single stranded)
Proteins
negatively charged
neutral (SDS added to give negative charge)

3. Components of Gel Electrophoresis:
Gel Matrix (agarose or polyacrylamide)
Electrophoresis Running Buffer (TAE or TBE)
Sample Loading Buffer
Gel Casting Tray with Comb
Gel Box
Power Supply
Sample (DNA, RNA, or protein)
Molecular Weight Marker/Ladder

4. Gel Matrix
Fibrous matrix that acts as a molecular strainer to aid in the separation of molecules based on:
Size
larger molecules separate from smaller molecules
Shape
linear molecules separate from globular molecules
Charge
positively charged molecules separate from negatively charged molecules

5. Gel Matrix Material 1) Agarose
Used in horizontal gel boxes to separate medium to large pieces of DNA and RNA
Carbohydrate derived from seaweed; non-toxic
Dissolved in a buffer solution (boiling required)
Usually TAE (Tris, Acetic Acid, EDTA) or TBE (Tris, Boric Acid, EDTA)

6. Gel Matrix Material 2) Polyacrylamide
Used in vertical gel boxes to separate smaller molecules such as proteins (mainly)
Rarely used to separate very small pieces of DNA or RNA
Cross-linked polymer of acrylamide; neurotoxin
Uses a Tris/Glycine/SDS buffer system

8. Agarose Gel Concentrations
Agarose can be used to separate …
DNA pieces between 500 and 25,000 base pairs (bp).
RNA pieces less than 5000 bases.
Higher concentrations of agarose result in a tighter mesh.
0.6 – 0.7%: used for very large DNA molecules
0.8 – 1.0%: most common; used for restriction digests and plasmids
2.0 – 3.0%: used for much smaller DNA fragments
Harder to prepare
Sometimes polyacrylamide is a better choice

9. Sample Movement based on SIZE
Small fragments move faster and farther from the well compared with larger fragments
Single stranded RNA moves faster through the gel than double stranded DNA
Fragments of similar size, charge, and shape will move at the same rate
Short molecules move through matrix easily; travels further
Large molecules require more effort to move through matrix; does not travel as far

10. Sample Movement based on CHARGE
A sample of charged molecules is loaded into the sample wells and power is added to establish an electrical field.
If the molecules have a net negative charge, they will move toward the anode (+ electrode: red).
If the molecules have a net positive charge, they will move toward the cathode (- electrode: black).

11. Sample Movement based on CHARGE
DNA and RNA are negatively charged due to their phosphate groups
Which direction will the DNA or RNA move?
They move toward the
positively charged anode!
RUNS TO RED!

12. Running Buffer Salt/Buffer solution:
Salt supplies the ions needed to establish an electric field.
Buffer stabilizes the pH to maintain the shape of the molecule being analyzed.
TAE (Tris, Acetic Acid, EDTA; pH ~ 8.2)
-- Used more often – cheaper
TBE (Tris, Boric Acid, EDTA; pH ~ 8.2)
Gels are usually run at V (~35 mAmps)

13. Sample Loading Buffer Contains: Glycerol:
Viscose chemical that adds density to sample so it sinks in well preventing the sample from floating out.
Tracking Dyes:
Adds color to sample so it can be seen to be loaded and tracked.
Xylene Cyanol -- mid size dye
Bromophenol Blue -- small size tracking dye (runs in front of DNA molecules)
Orange G -- very small dye; usually used with PCR
NOTE: these are NOT DNA stains

14. Gel Stains
DNA and RNA molecules are colorless therefore they need to be stained in order to be seen
Common Stains:
Methylene Blue
Ethidium Bromide (EtBr)
SybrSafe Green - NEW

15. Gel Stains Methylene Blue:
Stains DNA dark blue; visible under white light
Very low sensitivity; requires higher quantities of DNA to be visible
No health risks involved with its use; use in schools but not common in industry

16. Gel Stains Ethidium Bromide (EtBr): most commonly used stain
binds to DNA and fluoresces orange under UV light
extremely sensitive
TOXIC!!! carcinogenic and mutagenic with chronic exposure!
can be added to gel prior to casting or gel can be stained after the run.

17. binds to DNA and fluoresces green under UV light extremely sensitive
Gel Stains
SybrSafe Green:
New stain
binds to DNA and fluoresces green under UV light
extremely sensitive
SAFE --NOT TOXIC
SybrSafe Green vs Ethidium Bromide

18. Molecular Weight Marker/Ladder
A set of standards that are used to identify the approximate size of a molecules run on a gel during electrophoresis
contain fragments of known sizes
commercially purchased

19. Molecular Weight Marker/Ladder
Can you estimate the size of the DNA bands?