1. By: Tasnuva Jhileek Dr. Francine Norflus Biotechnology
Gel Electrophoresis
By: Tasnuva Jhileek
Dr. Francine Norflus
Biotechnology

2. What is Gel Electrophoresis?
A procedure that separates molecules based on size and charge.
Uses an electric field
Molecules move through gel made of agar or polyacrylamide

3. Simple Procedure
Molecules are dispensed into a well in the gel material.
Gel is placed in an electrophoresis apparatus
Electric current is applied to the contents of the apparatus
In the gel:
Larger molecules move more slowly
Smaller molecules move faster
The different sized molecules form distinct bands on the gel.

4. Types of Gel Agarose – DNA and protein
Polyacrylamide – Protein and DNA
Starch - protein

5. Agarose Gel Easy to cast, handle, store or dispose
Used to separate DNA fragments ranging from 50 base pair to several megabases (millions of bases).
Distance between DNA bands is determined by the percent agarose in the gel.
0.7% (large 5–10kb DNA fragments) and 2% (small 0.2–1kb fragments) agarose.
Low percentage gels - very weak and may break
High percentage gels - brittle and don’t set evenly

6. Polyacrylamide
used for separating proteins ranging in size from 5 to 2,000 kDa - Uniform pore size
Pore size is controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in creating a gel.
used to separate different proteins or isoforms of the same protein into separate bands and small DNA
gels are made in 6%, 8%, 10%, 12% or 15%.
percentage chosen depends on the size of the protein
smaller weight = higher gel

7. Starch Partially hydrolysed potato starch – protein electrophoresis
Non-denatured proteins are separated according to charge and size.
They are visualised using Napthal Black or Amido Black staining.
Typical starch gel concentrations are 5% to 10%

8. Nucleic Acid Electrophoresis
analytical technique used to separate DNA or RNA fragments by size.
an electric field induces the nucleic acids to migrate toward the anode
smaller fragments end up nearer to the anode
DNA is frequently cut into smaller fragments using a DNA restriction endonuclease (or restriction enzyme) or even PCR
Fragment size determination is typically done by comparison to commercially available DNA markers containing linear DNA fragments of known length
agarose (for relatively long DNA molecules) and polyacrylamide (for high resolution of short DNA molecules

9. Protein Electrophoresis
proteins, unlike nucleic acids, can have varying charges and complex shapes
proteins are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP)
the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.

10. Protein Electrophoresis cont.
Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.

11. Gel Condition
Denature - the native structure of macromolecules that are run within the gel is not maintained.
denatures the native structure of a protein
Native - separation method typically used in proteomics and metallomics.
does not use a charged denaturing agent.

12. Buffers
nucleic acids - Tris/Acetate/EDTA (TAE), Tris/Borate/EDTA (TBE).
TAE - lowest buffering capacity but best resolution for larger DNA. This means a lower voltage and more time, but a better product.
Xylene cyanol and Bromophenol blue are common dyes found in loading buffers

13. Visualization DNA may be visualized using ethidium bromide
SYBR Green I – more expensive, quicker, safer

14. Ethidium Bromide
light sensitive and is stored in a brown bottle covered in tin foil (aluminum foil).
fluoresces under UV light when intercalated into the major groove of DNA (or RNA)
any band containing more than ~20 ng DNA becomes distinctly visible
protein may be visualised using silver stain or Coomassie Brilliant Blue dye.
Mutagen
ALWAYS wear gloves

15. Areas of Application
used in forensics, molecular biology, genetics, microbiology and biochemistry.
results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device
the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel.
the measurement and analysis are mostly done with specialized software.

16. Lab Procedure Make Ethidium Bromide Make buffer
Add agarose to buffer and microwave
Pour gel after setting comb
Pour remaining buffer + EtBr in apparatus
Load gel (Black to Red 60 – 100 V)
Run gel
Kodak Moment 
Discard waste product safely

17. Making EtBr
1 g of Ethidium Bromide in 100 ml of dH20 in DARK colored bottle of transparent glass bottle wrapped in aluminum foil.
Dissolve completely (use Stir bar)
Use caution
Wear gloves at all times
Discard all EtBr related waste to biohazard

18. Making Buffer Calculation: Total of 300 ml of solution
15 ml of TBE ml dH2O

19. Making Gel Calculations: 0.8% agarose gel in 100 ml of solution
Mix 0.8 g of agarose gel in 100 ml of buffer solution.
Microwave until agarose dissolves completely in buffer.
Let it cool (not solidify).
Pour!

20. Preparing the DNA sample
10 microliter DNA + 2 microliter tracking dye in microcentrifuge tube.
Ladder: 1 microliter concentrated ladder per 9 microliter sterile water
Load all contents in separate wells in gel.

21. Setting up the apparatus
5 microliter Ethidium Bromide microliter TBE solution in electrophoresis apparatus
Plug apparatus into the electric outlet
Put gel in the apparatus (wells being in the black side)
DNA will travel from Black to Red
Set V to 60 or 100
Run DNA for about minutes

22. What is the safe way to discard the waste?
Put all EtBr related waste in the biohazard bin.
Discard buffer from the apparatus into biohazard bottle.
Wash gel in sink before throwing in the trash