Download DNA gel electrophoresis

DNA gel electrophoresis
Introduction
This technique is used for the separation of DNA on a matrix ( gel ) using electric
current.In this technique we can identify the concentration and the quality of the DNA
under study. Agarose gel that forms crosslinked polymer is used in this technique as a
matrix to separate different fragments of DNA. DNA fragments will appear on the gel
in different shapes and separates at different distances according to the type of the
DNA.
Electrophoresis equipments and supplies:
1-An electrophoresis chamber and power supply.
2-Gel casting trays:composed of UV-transparent plastic. They are different sizes and
shapes.
3-Sample combs: Are used to form sample wells in the gel.
4-Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA
(TBE).
5-Loading buffer, which contains something dense (e.g. glycerol or sucrose) to allow
the sample to "fall" into the sample wells, and one or two tracking dyes, to allow
visual monitoring or how far the electrophoresis has proceeded.
6-Ethidium bromide, a fluorescent dye used for staining nucleic acids.
7-UV Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium
bromide-stained DNA in gels.
Lab safty and precautions:
_ Ethidium bromide is a known mutagen and should be handled as a hazardous
chemical - wear gloves while handling.
Wear protective eyewear when observing DNA on a transilluminator to prevent
damage to the eyes from UV light.
Procedure:
1- Gel preparation
For 1% agarose add one gram agarose powder in 100 ml of the desired buffer.
The mixture should be heated on a hot plate until boiling so the agarose can dissolve
completely. Cool down the agarose mixture until 60 then pour off into a the casting
tray. Place the comb and let the gel solidify.
In the mean time prepare your sample.( add 10 ul of your sample + 10 ul loading
buffer and dye) mix thoroughly.
Once the gel is solid , remove the combs and familiarize yourself with the well
locations.
Plug in the Device . Make sure that the negative electrode is the closest to the loaded
DNA sample. Start the device and make sure that you observe the DNA movement on
the gel be tracking down the loading dye. If you forget this step the DNA will proceed
, leave the gel and lost in the buffer.
If the positive electrode was the closest to the loaded sample, what will happen?
DNA mobility rate on the gel:
1- .Type of DNA
There are 3 types: linear ( ds DNA) coiled and super-coiled (plasmids) and nicked
circles.( Fig 1). Fig. 1 represents the uncut and the cut form of the same plasmid.
Can you tell which migrate faster?
2- Agarose concentration
Uncut
cut
Fig. 1:
The corresponding figure shows the migration of the
same DNA marker onto three concentrations of agarose,
all of which were electrophoresed at the same voltage
and for identical times in the same gel tray.
1000 bp fragment is indicated in each lane.
At which agarose concentration this band had been
resolved the best?
Fig. 2.
3-Voltage: it is directly proportional with the mobility.
Yet, it is highly recommended to separate large fragments ( 4 Kb or more ) at lower
voltage to avoid ……………..
4-Electrophoresis Buffer: The most commonly used are TAE (Tris-acetate-EDTA)
and TBE (Tris-borate-EDTA). DNA fragments will migrate at different rates in these
two buffers due to differences in ionic strength. Buffers not only establish a pH, but
provide ions to support conductivity.
What will happen if you mistakenly use water instead of buffer?,
…………………………………………………………………….
…………………………………………………………………….
Conversely, if you use concentrated buffer (e.g. a 10X stock solution),
…………………………………………………………………………………………
……………………………………………………
Effects of Ethidium Bromide: Ethidium bromide intercalates between bases of
nucleic acids and allows very convenient detection of DNA fragments in gels. Thus,
binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its
mobility.
What will happen if you add too much ethidium bromide?
……………………………………………………………………………………….
Assignment:
1- To determine the size of the DNA we use a marker or a reference DNA as shown in
Fig 1. This is called a marker or a ladder. As you can see , it is composed of several
bands with a known size of base pairs.
M
Band 1
10000
8000
7000
6000
4000
2000
1000
750
500
1
2
3
4
Band 2
Band 3
According to the above example can you determine the size of bands 1, 2 & 3?
The size of band 1=
The size of band 2 =
The size of band 3=
2- Determine the type of isolated DNA in the following pictures?