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: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
: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
1
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
2
BIO 210L: Cells &Molecules Laboratory BIO 210L: DNA Extraction from Tissue
Using Salt Precipitation
:Result
3
BIO 210L: Cells &Molecules Laboratory BIO 210L: DNA Extraction from Tissue
Using Salt Precipitation
:Discussion
.Wells are holes that hold DNA samples•
4
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