Transcript الشريحة 1

Purposes
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•
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1.
2.
To understand the principle of Gel
electrophoresis.
To become familiar with the part of the
electrophoresis setup.
Electrophoresis is a laboratory technique for
separating molecules based on their charge and
or size.
This technique is simple:
Biological samples are prepared and placed in
gel (matrix).
Electricity is then run through the matrix,
causing molecules in the samples to separate.
There are two components of electrophoresis
system:
1.
2.
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Electrical filed.
The physical resistance of the matrix.
Electrical filed
Many molecules (amino acids, proteins, DNA, and RNA)
have naturally occurring negative and positive charges on
them.
The sum of these charges determines the overall charge.
At neutral pH:
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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.
Separation of a Mixture of Charged
Molecules
Charged molecules are separated based on their electrical charge
and size.
Positive Molecules
Analyze
Charge
Separation
Size
Separation
Mixture of
Charged Molecules
Identify
Purify
Negative Molecules
 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.
• This barrier acts as a ”sieve” to separate the molecules by size.
• Without any resistance to there movement, “equally charged
molecules will migrate through the electrical field at the same
rate.
• If we put barrier in there way small molecules can move more
quickly than large one.
Molecule Size:
• The molecule suspended in a gel.
• The microscopic particles attach to one another forming
tunnels that act as a sieve to separate the molecules.
• Small molecules can move faster than large molecules.
• porous material is made of microscopic particles
Porous
Material
Proteins Entering
Porous Material
Smallest Move
Fastest
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
Cathode
Anode
+
Buffer
Dyes
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.
•
Polyacrylamide “chain of acrylamide molecules”.
– It is often used to make plastics and rubber.
– It has a small pore size good for separating small molecules.
Agarose Gel
• A porous material derived from red seaweed
• 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.
1% agarose
• Concentration affects molecules migration :• Low conc. = larger pores better
resolution of larger DNA fragments
• High conc. = smaller pores better
resolution of smaller DNA fragments
2% agarose
Fragment Resolution
Most agarose gels are made with between 0.7% (good separation
or resolution of large 5–10kb DNA fragments) and 2% (good
resolution for small 0.2–1kb fragments) agarose dissolved in
electrophoresis buffer. Up to 3% can be used for separating very
tiny fragments but a vertical polyacrylamide gel is more
appropriate in this case. Low percentage gels are very weak and
may break when you try to lift them. High percentage gels are
often brittle and do not set evenly. 1% gels are common for many
applications.
% Agarose
DNA fragment, kb
0.5
30-1
0.7
12-0.8
1.0
10-0.5
1.2
7-0.4
1.5
3-0.2
• Agarose at room temperature is a 3-dimentional solid
matrix.
• The smaller the fragments the further the migration or
movement through the matrix.
small
large
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Power
+
Electrophoresis Buffer
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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”.
The important feature of any buffer is that it contains an electrolyte so
that it can conduct electricity.
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Concentration affects DNA migration
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Use of water will produce no migraton
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High buffer conc. could melt the agarose gel
Purposes for Agarose Gel Electrophoresis
• Analysis of molecules size.
• Separation and extraction of molecules.
• Quantification of molecules.
Overview of Agarose Gel Electrophoresis
• Gel Preparation
• Loading the gel
• Running the gel
Gel Preparation
Agarose is a linear polymer extracted from seaweed.
Agarose
Buffer Solution
Combine the agarose powder and buffer solution. Use a flask that is
several times larger than the volume of buffer.
Melting the Agarose
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.
Loading the gel
Running the gel