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BIO 210L: Cells &Molecules Laboratory BIO 210L: DNA Extraction from Tissue

Using Salt Precipitation

:Introduction
Electrophoresis is a technique used in the laboratory that results in the separation of
charged molecules. DNA is a negatively charged molecule, and is moved by electric current
.through a matrix of aragose

Aragose gel electrophoresis is the easiest and commonest way of separation and
visualizing of DNA fragments. The fragments are separated by size by forcing them to move
through an agarose gel matrix, which is subjected to an electric field. The electric field is
.(generated by applying potential (voltage) across an electrolytic solution (buffer

Agarose (extracted from seaweed) is a linear polymer composed of alternating


residues of D- and L-galactose joined by α (1 3) and β (1 4) glycositic linkage. Purified
agarose is in the powdered form, and is insoluble in water (or buffer) at room temperature,
however, it dissolves in boiling water . when it starts to cool, it undergoes what is known as
polymerization. Rather than staying dissolved in the water or coming out of solution, the
sugar polymers crosslink with each other, causing the solution to "gel" into a semi-solid
.matrix much like "Jello" only more firm

:Materials Required
DNA fragment•
10x loading buffer•
Electrophoresis buffer•
(1x TBE (Tris Borate EDTA buffer•
Bromophenol Blue•
Sodium Dodecyl Sulfate•
Ethedium Bromide•
Equipment for Agarose gel electrophoresis•
UV – light source•
:Method
:(Preparing 50ml of 1% agarose (a seaweed derivative
.Weigh 0.5g of agarsoe powder using a weighing paper and mettle balance-1
.Put the powder in an Erlenmeyer flask and add 50ml of buffer-2
Heat in the microwave for couple minutes (be careful not to overheat it, it-3
(might become superheated and may boil violently
.Swirl gently to allow all the grains of agarose to dissolve-4
.Note: the buffer is used to maintain a certain PH•
Note: Agarose gel is used to slow the movement of DNA and separate it by•
size. Also when we increase the concentration of the agarose we make the
agarose pores smaller thus making it harder for DNA to migrate or if it is
.too concentrated inhibiting it permanently
Pour the warm agarose quickly because it solidifies when it becomes cold.•
.Pouring it warm prevent it from solidifying in the Erlenmeyer flask

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BIO 210L: Cells &Molecules Laboratory BIO 210L: DNA Extraction from Tissue
Using Salt Precipitation

Mettler balance

Pouring agarose

:Procedure
Gentl pour the warm agarose solution into the plate with casting combs in-1
.place
Check that no air bubbles are under or between the teeth of the comb. Air-2
bubbles present in the molten gel can be removed easily by poking them
.with a clean pipette tip
.Allow the gel to set completely for 34 – 45 minutes at room temperature-3
Pour small amount of electrophoresis buffer on top of the gel, and -4
.carefully remove the comb
Add just enough electrophoresis buffer to cover the gel depth of about -5
.1nm
.Load the first well with marker-6

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BIO 210L: Cells &Molecules Laboratory BIO 210L: DNA Extraction from Tissue
Using Salt Precipitation

To prepare samples for electrophoresis, usually the lab concentration is-7


10x more concentrated than the ordinary. We need to get 20µl sample of
:1x concentration so we apply the following equation
C1V1 = C2V2  10x V1 = (1x)(20µl)  V1 = 2µl
Then we put 2µl of loading buffer and 18 µl of DNA. This changes the DNA
color making it visible in the gel and increase its density to let it rest in the
well (the loading buffer contains bromophenol blue for color and glycerol for
.(increasing the density of the DNA
Add 5% Ethidium Bromide (in the 50µl we need 2.5µl) to make the DNA -8
fluorescence. (be careful Ethidium Bromide is highly carcinogenic, so
.(wear gloves at all times
Load the rest of the wells with 20µl samples. Be careful not to break the-9
.wells. If your hand shakes then use your other hand as a stage
Close the gel tank, switch on the power source and run at 100 volts until -10
dye marker have migrated and appropriate distance, depending on the size
of DNA. (pay attention to the migration process so not to lose the samples
.(if it migrate too much
.Monitor the progress of the gel by reference to the marker dye-11
Switch off and unplug the gel tank and carry the gel to the darkroom to-12
look at the UV light box. (Be aware not to look directly to the UV light as
.(it is carcinogenic and to avoid vision injury
.

The right way to load the wells

:Result
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BIO 210L: Cells &Molecules Laboratory BIO 210L: DNA Extraction from Tissue
Using Salt Precipitation

.DNA electrophoresis shows the DNA fractions

Group one DNA agarose electrophoresis

DNA agarose electrophoresis done in a specialist lab

:Discussion
.Wells are holes that hold DNA samples•
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BIO 210L: Cells &Molecules Laboratory BIO 210L: DNA Extraction from Tissue
Using Salt Precipitation

Be careful when laying the agarose in the casting tray making sure that the •
wells are on the negative side to avoid the migration of the DNA fragments
the wrong way and losing them in the solution as the DNA fragments are
.negatively charged moving away from the negative end to the positive end
Bubbles can be seen as a sign of successful electric flow through the •
.casting tray
The comb creates the wells in the agarose gel which consequently is used •
.as a reservoir to hold DNA sample
The DNA migration speed depends on the strength of the electrical field,•
buffer concentration, density of agarose, and the size of DNA.(small DNa
.(fragments move faster than the bigger ones
The agarose gel mix acts as a sieve for DNA molecules. The smaller the•
DNA fragment the easier to move through the holes thus giving the
.lagging DNA shape
Molecular weight markers are used to estimate the size of DNA fragments•
.in the DNA sample
Place the cover on the electrophoresis chamber to avoid any splash by•
mistake and connect the electrical leads correctly to avoid mixing the poles
and the migration of the DNA to the opposite way resulting in losing the
.DNA in the process

:Acknowledgement
I would like to thank Mrs. Ansar El Andari for her instructions and my group
.for their help

:Literature Cited

.Lab supplement provided by the lab instructor Ms. Ansar El Andari•


http://www.steve.gb.com/images/science/agarose_gel.jpg•