History[ edit ] The term plasmid was introduced in by the American molecular biologist Joshua Lederberg to refer to "any extrachromosomal hereditary determinant.
Pei Yun Lee at ude. To view a copy of this license, visit http: Abstract Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from bp to 25 kb1.
Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose L- and D-galactose subunits2. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size.
To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA and RNA molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode.
The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye.
By following this protocol, students should be able to: Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3.
Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5.
Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments Keywords: Preparation of the Gel Weigh out the appropriate mass of agarose into an Erlenmeyer flask.
The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0. Add running buffer to the agarose-containing flask. This is most commonly done by heating in a microwave, but can also be done over a Bunsen flame.
At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved. Add ethidium bromide EtBr to a concentration of 0. Alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.
EtBr is a suspected carcinogen and must be properly disposed of per institution regulations. Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the staining of DNA are available; however EtBr remains the most popular one due to its sensitivity and cost.
Failure to do so will warp the gel tray.
Place the gel tray into the casting apparatus. Alternatively, one may also tape the open edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells. Pour the molten agarose into the gel mold. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box.
Gel loading dye is typically made at 6X concentration 0. Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel. Add enough running buffer to cover the surface of the gel. It is important to use the same running buffer as the one used to prepare the gel.
Attach the leads of the gel box to the power supply. Turn on the power supply and verify that both gel box and power supply are working. Slowly and carefully load the DNA sample s into the gel Fig.Gel electrophoresis using agarose, a highly purified linear polysaccharide derived from agar, has been widely used in the detection and characterization of plasmids, also the linear DNA fragments.
o Explain how electrophoresis of DNA works.
o Explain how electrophoresis can be used to determine the size of a fragment of DNA. I. Objectives: • Isolate a plasmid containing a cloned soybean gene. • Use restriction enzymes to release the soybean gene from the plasmid. • Use gel electrophoresis to determine the size of the soybean gene.
LabBench Activity Plasmids. Plasmids are circular pieces of DNA that exist outside the main bacterial chromosome and carry their own genes for specialized functions.
In genetic engineering, plasmids are one means used to introduce foreign genes into a bacterial cell. To understand how this might work, consider the plasmid below. An experienced lab report writer should be well trained and have done the gel electrophoresis of DNA experiment in order to deliver top-notch gel electrophoresis lab report.
Testimonials I was impressed by Custom Papers Writing Help with their amazing guidelines on action research pape r. Agarose gel electrophoresis (discussed also in Chapter 7) is the most commonly used method for the size- and shape-based separation of DNA molecules comprising several hundred or more base pairs, including plasmid DNA molecules (Figure ).
Analysis of Plasmid DNA By Restriction Digestion and Agarose Gel Electrophoresis This experiment will provide an introduction to the use of restriction enzymes and gel electrophoresis, the two most fundamental techniques in recombinant DNA methodology.
You will learn how to cut DNA with a restriction endonuclease and analyze the .