Transcript lab9

DNA Gel Electrophoresis
-Gel electrophoresis detects the presence of DNA in a sample.
-Gel electrophoresis detects the number of nucleotides in a fragment of DNA
This procedure depends on :
•
the nucleic acid charge and the used solution polarity. DNA molecules are
negative (-) in charge. When DNA exposed to electrical current, the
negatively charged DNA pieces will move and migrate to the positive (+)
pole depending on their molecular weight.
•
Electrical migration takes place in a gel rather than free solutions.
Factors Affecting the Sample Migration:
1- Size, form and type of charge.
2- Strength of the electrical field.
3- pH of the buffer.
4- Concentrations of solutions.
Purpose of Gel Electrophoresis
•
•
A method for separating DNA
Can be used to separate the size of
– DNA
– RNA
– Protein
How It Separates
The gel is a porous matrix (like a sponge)
Separates DNA based on
-Size
-Charge
Separation by Size
• As DNA is moved through the
gel, smaller sized fragments
move through faster than
larger sized fragments
– Ex. A 100 base pair fragment
will move through the gel faster
than a 500 bp fragment
start
end
Separation Using Charge
•
•
•
The charge on DNA is what makes it move
through the gel
DNA is a charged molecule. What is the charge
on DNA?
– Negative charge
Why?
– Phosphate group is negatively charged
Concentrations of solutions
- Higher concentrations of agarose facilitate
separation of small DNAs, while low agarose
concentrations allow resolution of larger DNAs.
– Low conc. = larger pores better resolution of
larger DNA fragments
– High conc. = smaller pores better resolution of
smaller DNA fragments
1% agarose
2% agarose
Used gels are of two kinds:
1- Agarose gel: Extracted from sea weed (algae).
Agarose gel electrophoresis is more efficient in
isolating large nucleic acid pieces (from 1-60 Kb or
more). While
2-Polyacrylamide gel: Artificial gel. Polyacrylamide
gel electrophoresis (PAGE) is used to isolate smaller
pieces ( from 1Kb and less).
A- Components of an Electrophoresis System
• Power supply and chamber, a source of negatively charged particles with a
cathode and anode
• Buffer, a fluid mixture of water and ions The most commonly used for duplex DNA
are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). Buffers not only establish
a pH, but provide ions to support conductivity. Concentration affects DNA migration
– Use of water will produce no migraton
– High buffer conc. could melt the agarose gel
– New Sodium Borate (SB) buffer allows gels to be run at higher voltages in less
time than traditional buffers
• Agarose gel, a porous material that DNA migrates through
• Gel casting materials
• DNA ladder, mixture of DNA fragments of known lengths
• Loading dye, contains a dense material such as glycerol, to allow the sample to
"fall" into the sample wells and allows visualization of DNA migration
• DNA Stain, allows visualizations of DNA fragments after electrophoresis (Ethidium
bromide) binds to DNA and fluoresces under UV light, allowing the visualization of
DNA on a Gel.
B- Required Tools for the Procedure:
•Micropipetteor rang 0.5-20 μl and Yellow tips.
•Electrophoresis apparatus
•UV Transilluminator
•Digital camera
•Conical flask
•Boling water bath or Microwave oven
•Microcentrifuge tube (Eppendorf tube)
•Parafilm
•Aluminum foil
•Electrophoresis apparatus:
•Tank:
It is the container which contain the buffer. It always has a cover to prevent the
evaporation of buffer and for safety. There are two types of tanks: one is called the
horizontal while the other is the vertical. In the horizontal tank we are using
agarose or acrylamide gel as a support media and it is used to identify DNA and
RNA. On the other hand, vertical tank only polyacrylamide gel is used. Vertical is
used mainly in identifying protein and in DNA sequencing.
•Tray: Is the actual mold which provides a shape for the gel as it polymerizes (or
solidify). After polymerization, the gel will move out of the mold and submerse it
in a tank of buffer to run.
•Support: This is just a small piece of glass or plastic that rests snugly in the
bottom of the tray. When the gel is finished polymerizing, the support is gently
pushed upwards out of the tray.
•Power supply: It can be monitored and operated in current (amps), voltage
(volts) or power (watts) mode. The black and red cords leading from the power
supply are then attached to the tray in which the DNA in the gel is run.
•Combs: It used to make wells on the gel to load different samples.
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Cathode
Anode
Buffer
Dyes
Agarose gel
+
Power Supply
Materials:
Agarose powder. TBE buffer.
Ethidium bromide (10mg/ml).
6X Loading buffer (bromophenol blue 0.25% (w/v) and sucrose 40% (w/v)(.
Samples (DNA sample, PCR products, restricted (cut)PCR products ).
Buffer preparation:
10X TBE buffer
Make 0.89 M Tris-Base (108 g/L), 0.89 M Boric acid (55.0 g/L), and 0.02 M (7.44 g/L)
of EDTA-Na2-salt, complete to 1000 ml with distilled water, and then adjust pH to
8.3 (with NaOH).
1X TBE buffer
Take 100 ml form 10X stock solution and complete to 1000 ml with distilled water.
Then fill the tank of electrophoresis and dissolve agarose powder with this buffer.
Gel preparation:
•Dissolve agarose powder (2%), i.e. 2 gm in 100 ml 1X TBE buffer.
•Melt the gel in a microwave oven or in boiling water bath until completely melted.
•Cool the solution to about 60°C.
•Add 1 µl ethidium bromide stock per 10 ml gel solution and mix gently.
1-Weigh out a gram of
agarose.
Agarose
Buffer Solution
2-Mix the agarose with 50100 ml of buffer.
Gel is prepared by dissolving specific amount of used gel in gel buffer Use a flask that is
several times larger than the volume of buffer
•Agarose is insoluble at room temperature (left).
•The agarose solution is boiled until clear (right).
3- Heat to dissolve the agarose.
4-
Assemble the gel tray and comb.
5- Pour the gel.
6- Gel solution is allowed to cool and solidify
7- Loading Dye with
DNA Stain
8- Loading the samples
The Gel
Wells are created to put the DNA into
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wells
-
well
Direction DNA travels
+
Direction
DNA
travels
SIDE VIEW
TOP VIEW
+
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Turn the switch on the power supply to "Off" before connecting the electrophoresis
chamber.
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Place the lid tightly on the chamber and plug the electrical leads into the recessed output
jacks of the power supply.
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Plug the red (+) lead into the red jack, and the black (-) lead into the black jack. As the
voltage applied to a gel is increased,
•
larger fragments migrate proportionally faster that small fragments. For that reason,
the best resolution of fragments larger than about 2 kb is attained by applying no more
than 5 volts per cm to the gel (the cm value is the distance between the two
electrodes, not the length of the gel).
Power supply
DNA can be visualized by exposing the gel to UV
transilluminator**. When using agarose gel, DNA
appears as orange bands. And when using polyacrylamide
gel appears as black bands with silver nitrate stain.
Unknown DNA fragments sizes are estimated by reference
indices.
* Ethidium bromide is mutagen and should be handled as a
hazardous chemical, wear gloves when handling.
** Always wear protective eyewear when observing DNA on a
transilluminator to prevent damage to the eyes from UV light.
Relative Size vs. Absolute
Size
• Looking at a gel, you can
determine which fragments
of DNA are bigger than
others = Relative Size
• Which fragment is bigger,
A or B?
– Fragment A (didn’t travel as
far in a fixed amount of
time)
(-) start
A
B
(+) end
Absolute Size
• How can we determine the actual size
of the DNA fragments (how many base
pairs- bp)?
• Use a size standard
– Also called a DNA ladder
– Consists of a series of fragments of known
sizes
– Use it to compare to your DNA fragments
Example
• Suppose you have a
size standard with the
following sized
fragments: 1000 bp,
850 bp, 750 bp, 600
bp, 200 bp, 100 bp
• Based of this info,
how big is the
circled fragment?
–850 bp
1000 bp
850 bp
750 bp
600 bp
100 bp