Gel Elpho

Gel Electrophoresis
Gel Electrophoresis
Polyacrylamide Gel electrophoresis is a widely used
technique for the analysis of nucleic acids and
proteins. Agarose gel electrophoresis is routinely used
for the preparation and analysis of DNA.
Gel electrophoresis is a procedure that separates
molecules on the basis of their rate of movement
through a gel under the influence of an electrical
field.
How Separation Occurs

Electrical Charge:
Many molecules (amino acids, peptides, proteins, DNA, and RNA)

have naturally occurring negative and positive charges on them.
The sum of these charges determines the overall charge.
When introduced to an electrical current, negatively charged
molecules are attracted to the positive electrode and positively
charged molecules are attracted to the negative electrode.
+
+
N
Positively
Charged
- +
+ - + - + +
- +
+
+
+
Positively Charged
Peptide
Negatively Charged
Protein
DNA is negatively charged.

When placed in an electrical field, DNA will migrate toward the positive
pole (anode).
An agarose gel is used to slow the movement of DNA and separate by size.
O2
DNA
Power
Polymerized agarose is porous,

allowing for the movement of DNA
Scanning Electron Micrograph
of Agarose Gel (11 m)
How Separation Occurs

Molecule Size:
The porous material is made of microscopic particles
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
Proteins
Entering
Porous Material
Smallest
Move Fastest
Gel Electrophoresis
Gels can be made from substances such as
agarose or polyacrylamide.
Agarose a complex sugar chain from red
seaweed. It is commonly used in foods (ice
cream, whipped cream, and jellies) and many
biological mediums. It has a large pore size
good for separating large molecules quickly.
Polyacrylamide chain of acrylic acid
Red Sea
Weed
molecules. It is often used to make plastics and

rubber. It has a small pore size good for
separating small molecules slowly.
*Polyacrylamide is a neurotoxin!
Acrylic Acid
How fast will the DNA migrate?

strength of the electrical field, buffer, density of agarose gel
Size of the DNA!
*Small DNA move faster than large DNA
gel electrophoresis separates DNA according to size
DNA
small
Power
large
Within an agarose gel, linear DNA migrate inversely

proportional to the log10 of their molecular weight.
Making an Agarose Gel
An agarose gel is prepared

by combining agarose
powder and a buffer
solution.
Buffer
Flask for boiling
Agarose
Electrophoresis Equipment
Power supply
Cover
Gel tank
Electrical leads
Casting tray
Gel combs
Gel casting tray & combs
Pouring the gel
Allow the agarose solution to cool slightly (~60C) and then carefully
pour the melted agarose solution into the casting tray. Avoid air
bubbles.
Each of the gel combs should be submerged in the melted agarose solution.
When cooled, the agarose polymerizes, forming a flexible gel. It should

appear lighter in color when completely cooled (30-45 minutes).
Carefully remove the combs and tape.
Place the gel in the electrophoresis chamber.
DNA
buffer
wells
Cathode
(negative)
Anode
(positive)
Add enough electrophoresis buffer to cover the gel to a depth of

at least 1 mm. Make sure each well is filled with buffer.
Sample Preparation
Mix the samples of DNA with the 6X sample loading buffer (w/ tracking
dye). This allows the samples to be seen when loading onto the gel, and
increases the density of the samples, causing them to sink into the gel
wells.
6X Loading Buffer:
Bromophenol Blue (for color)
Glycerol (for weight)
Loading the Gel
Carefully place the pipette tip over a well and gently expel the sample.
The sample should sink into the well. Be careful not to puncture the
gel with the pipette tip.
Running the Gel
Place the cover on the electrophoresis chamber, connecting the electrical

leads. Connect the electrical leads to the power supply. Be sure the leads
are attached correctly - DNA migrates toward the anode (red). When the
power is turned on, bubbles should form on the electrodes in the
electrophoresis chamber.
Cathode
(-)
wells
Bromophenol Blue
DNA
(-)
Gel
Anode
(+)
After the current is applied, make sure the Gel is running in the correct
direction. Bromophenol blue will run in the same direction as the DNA.
DNA Ladder Standard

12,000 bp
5,000
DNA
migration
Note: bromophenol
blue migrates at
approximately the
same rate as a 300 bp
DNA molecule
bromophenol blue
2,000
1,650
1,000
850
650
500
400
300
200
100
+
Inclusion of a DNA ladder (DNAs of know sizes) on the gel
makes it easy to determine the sizes of unknown DNAs.
Staining the Gel

Ethidium bromide binds to DNA and fluoresces under UV light,
allowing the visualization of DNA on a Gel.
Ethidium bromide can be added to the gel and/or running buffer
before the gel is run or the gel can be stained after it has run.
***CAUTION! Ethidium bromide is a powerful mutagen and is

moderately toxic. Gloves should be worn at all times.
Safer alternatives to Ethidium Bromide

Methylene Blue
BioRAD - Bio-Safe DNA Stain
Wards - QUIKView DNA Stain
Carolina BLU Stain
others
advantages
Inexpensive
Less toxic
No UV light required
No hazardous waste disposal
disadvantages
Less sensitive
More DNA needed on gel
Longer staining/destaining time
Staining the Gel
Place the gel in the staining tray containing warm diluted stain.
Allow the gel to stain for 25-30 minutes.
To remove excess stain, allow the gel to destain in water.
Replace water several times for efficient destain.
Ethidium Bromide requires an ultraviolet light source to visualize
Visualizing the DNA (ethidium bromide)

DNA ladder
1
DNA ladder
8
wells
5,000 bp
2,000
1,650
1,000
850
650
500
400
300
200
100
PCR Product
Primer dimers
Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA
Visualizing the DNA (QuikVIEW stain)
wells
DNA ladder
PCR
Product
+ - - - - + + - - + - +
2,000 bp
1,500
1,000
750
500
250
Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA
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Gel Electrophoresis

How Separation Occurs

Many molecules (amino acids, peptides, proteins, DNA, and RNA)

DNA is negatively charged.

Polymerized agarose is porous,

How Separation Occurs

Polyacrylamide chain of acrylic acid

molecules. It is often used to make plastics and

How fast will the DNA migrate?

Within an agarose gel, linear DNA migrate inversely

Making an Agarose Gel

An agarose gel is prepared

Flask for boiling

Gel casting tray & combs

Pouring the gel

When cooled, the agarose polymerizes, forming a flexible gel. It should

Place the gel in the electrophoresis chamber.

Add enough electrophoresis buffer to cover the gel to a depth of

Loading the Gel

Running the Gel

Place the cover on the electrophoresis chamber, connecting the electrical

DNA Ladder Standard

Staining the Gel

***CAUTION! Ethidium bromide is a powerful mutagen and is

Safer alternatives to Ethidium Bromide

Staining the Gel

Ethidium Bromide requires an ultraviolet light source to visualize

Visualizing the DNA (ethidium bromide)

Samples # 1, 4, 6 & 7 were positive for Wolbachia DNA

Visualizing the DNA (QuikVIEW stain)

Samples # 1, 6, 7, 10 & 12 were positive for Wolbachia DNA

Is this man the father of the child?

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