Scott Malcolm is a great businessman, who lives in Dallas, Taxes. He explains here about biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length and to estimate the size of  DNA and RNA fragments.

1. Gel electrophoresis
By:- Scott Malcolm

2. Purposes
• To understand the principle of Gel electrophoresis.
• To become familiar with the part of the electrophoresis setup.
• Gel electrophoresis: is a method used in
• Clinical chemistry to separate proteins by charge and or
size.
• Biochemistry and molecular biology to separate a mixed
population of DNA and RNA fragments by length and to
estimate the size of DNA and RNA fragments.

3. • Nucleic acid molecules are separated by applying an electric
field to move the negatively charged molecules through
an agarose matrix.
• Shorter molecules move faster and migrate farther than longer
ones because shorter molecules migrate more easily through the
pores of the gel.
• This phenomenon is called sieving.

4. Applications of Agarose Gel
Electrophoresis
• Separation of restriction enzyme digested DNA including
genomic DNA, prior to Southern Blot transfer. It is also often
used for separating RNA prior to Northern transfer.
• Analysis of PCR products after polymerase chain reaction to
assess for target DNA amplification.
• Allows for the estimation of the size of DNA molecules using a
DNA marker or ladder which contains DNA fragments of various
known sizes.
• Allows the rough estimation of DNA quantity and quality.

5. There are two components of electrophoresis
system:
1. Electrical filed.
2. The physical resistance of the matrix.
 Electrical filed
• The sum of these charges determines the overall charge.
• At neutral pH:
 proteins have a unique electrical charged.
 both DNA and RNA bases are negative charged.
• Molecules with a negative charge (anions) will be attracted
to the positively charged node (anode)……. Red color.
• Molecules with a positive charge (cations) will be attracted
to the negatively charged node (cathode)…… Black color.

6. Charged molecules are separated based on their electrical charge
and size.
Separation of a Mixture of Charged
Molecules
Charge
Separation
Size
Separation
Analyze
Identify
PurifyMixture of
Charged Molecules
Positive Molecules
Negative Molecules

7. The physical resistance of the matrix
• Electrophoresis is conducted through a gel substances
called a matrix.
• Matrix acts as a physical barrier to movement of the
substances.
• Without any resistance to there movement, “equally
charged molecules will migrate through the electrical
field at the same rate.

8. Molecule Size
Porous
Material
Proteins Entering
Porous Material
Smallest Move
Fastest

9. Components of an Electrophoresis System
1. Power supply and chamber: a source of power supply
2. Agarose gel: a porous material that molecules migrates
through
3. Buffer: a fluid mixture of water and ions.
4. Gel casting materials
Power Supply

10. Buffer
Dyes
+
-
Cathode
Anode

11. Gel Electrophoresis
• They are many types of matrices used in electrophoresis, but all
function similarly as physical barrier to movement.
Gels can be made from substances such as agarose or
polyacrylamide.
• Agarose “ a complex sugar chain from red seaweed”.
• Non toxic carbohydrate.
• It is commonly used in foods (ice cream, and jellies)
and many biological mediums.
• It has a large pore size good for separating large
molecules quickly.

12. Agarose Gel
• Agarose is highly purified to remove impurities
and charge.
• Acts as a sieve for separating molecules.
• This solid matrix will allow the separation of
fragments by size.
• Concentration affects molecules migration :-
• Low conc. = larger pores better
resolution of larger DNA fragments
• High conc. = smaller pores better
resolution of smaller DNA fragments
1% agarose
2% agarose

13. Electrophoresis Buffer
• TAE (Tris -acetate-EDTA) and TBE (Tris-borate-EDTA) –
pH buffer.
1. Tris a pH buffer.
2. Acetic acid provide ions to support conductivity and
maintain pH.
3. EDTA, prevent brake down of molecules. “all dissolved
in water”.
 Concentration affects DNA migration
• Use of water will produce no migraton
• High buffer conc. could melt the agarose gel

14. • Gel Preparation
• Loading the gel
• Running the gel
Overview of Agarose Gel Electrophoresis

15. Agarose is a linear polymer extracted from seaweed.
Gel Preparation

16. Agarose Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.

17. Agarose is insoluble at room temperature (left).
The agarose solution is boiled until clear (right).
Gently swirl the solution periodically when heating to allow all the grains of agarose to
dissolve.
***Be careful when boiling - the agarose solution may become superheated and may boil
violently if it has been heated too long in a microwave oven.
Melting the Agarose

20. Loading the gel

21. • Gels are covered with a buffer solution. Prior to
loading the samples.
• The DNA must be mixed with a loading dye.
• The loading dye serves two purposes:
1. Increases the density of the DNA so it will sink into the wells.
2. Provides a visual marker so you know how far the DNA (which is
not visible) has traveled in the gel.

23. Running the gel

24. Visualization
• DNA may be visualized using ethidium
bromide which, when intercalated into
DNA, fluoresce under ultraviolet light.
• While protein may be visualised using silver stain
or Coomassie Brilliant Blue dye.

26. What is Wrong with Agarose Gels?
• Poor precision
• Low sensitivity
• Low resolution
• Non-automated
• Results are not expressed as numbers
• Ethidium bromide staining is not very
quantitative

27. Sodium DodecylSulphate- PolyAcrylamide
Gel Electrophoresis

28. • Polyacrylamide is a cross-linked polymer of acrylamide.
• Have smaller pores than agarose, therefore high degree of
resolving power.
• Can separate DNA fragments which range in size from
10-500 bp.
• DNA fragments which differ in size by one nucleotide can
be separated from each other.

29. Protein Electrophoresis
• Proteins, unlike DNA, do not have a constant size to
charge ratio
• In an electric field, some will move to the positive and some to the
negative pole, and some will not move because they are neutral.
• The purpose of this method is:
• Separate proteins according to their size, and no other
physical feature. In order to understand how this works,
we have to understand the two halves of the name: SDS
and PAGE

30. Principle
• We need a way to convert all proteins to the same shape - we use
SDS.
• SDS (sodium dodecyl sulfate) is a detergent that can dissolve
hydrophobic molecules but also has a negative charge (sulfate)
attached to it.
• The end result has two important features:
1.all proteins contain only primary structure and
2.all proteins have a large negative charge which means they
will all migrate towards the positive pole when placed in an
electric field.
• They migrate through a gel towards the positive pole at a rate
proportional to their linear size

31. Now we are ready to focus on the second half - PAGE

32. • PAGE is the preferred method for
separation of proteins.
• Polymerization of acrylamide is
initiated by the addition of
TEMED.
• Bis-Acrylamide polymerizes along
with acrylamide forming cross-
links between acrylamide chains

33. Movement of Proteins in Gel
• smaller proteins will move
through the gel faster while larger
proteins move at a slower pace.
bands.
• These can be transferred onto a
nitrocellulose to be probed with
antibodies and corresponding
markers, such as in a western blot.

34. Vertical Gel Format: Polyacrylamide Gel Electrophoresis

35. Step by Step Instructions on how to assemble the polyacrylamide
gel apparatus

37. Sample Buffer
• Tris buffer to provide appropriate pH
• SDS (sodium dodecyl sulphate) detergent to
dissolve proteins and give them a negative
charge
• Glycerol to make samples sink into wells
• Bromophenol Blue dye to visualize samples

38. Loading Samples & Running the gel

40. Thanks