Alkaline lyses method was used for the isolation of plasmid from bacterial cells. The method used was as described in cloning manual (Sambrook et al., 1989). Plasmid minipreps were made from 1.5 ml of overnight culture and Briefly, cells were pelleted and cell pellets from 1.5 ml cultures were resuspended in 100 μl of Glucose-Tris-EDTA (GTE) solution (Glucose 50 mM, Tris pH 7.5, 25 mM, EDTA 10 mM), incubated on ice followed by lysis in 200 μl of a solution containing 0.2N NaOH and 1% Sodium dodecyl sulphate (SDS) and mixed by gentle inversion. The cell lysates were treated with 150 μl of 5M potassium acetate (pH 5.8) on ice and then centrifuged at 13000 rpm for 10 min. The supernatants were removed and DNA was precipitated with 800 μl of ethanol. Followed by centrifugation. Isolated plasmid DNA was then washed with 75% alcohol and air-dried. The pellet was resuspended in 30 μl of autoclaved distilled water water containing 10 μg/ml RNAse A.
AGAROSE GEL ELECTROPHORESIS
Agarose gel electrophoresis is a method used in biochemistry and molecular biology to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate farther than longer ones The most common dye used to make Agarose gel electrophoresis can be used for the separation of DNA fragments ranging from 50 base pair to several megabases (millions of bases) using specialized apparatus. However, it is normally used in a range of 100 bp to 20 kbp on an approximately 1% gel. Typical run times are about an hour.
Small nucleic acids are better separated by polyacrylamide gels, large DNA molecules are only able to move end-on in a process called "reptation" and are more difficult to separate. In general lower concentrations of agarose are better for larger molecules; it will exaggerate the distances between bands. The disadvantage of higher concentrations is the long run times (sometimes days). Instead these gels should be run with a pulsed field electrophoresis (PFE), or field inversion electrophoresis.DNA or RNA bands visible for agarose gel electrophoresis is ethidium bromide, usually abbreviated as EtBr. It fluoresces under UV light when intercalated into DNA (or RNA). By running DNA through an EtBr-treated gel and visualizing it with UV light, distinct bands of DNA become visible. EtBr is a known carcinogen, however, and safer alternatives are available.
SYBR Green I is another dsDNA stain, produced by Invitrogen. It is more expensive, but 25 times more sensitive, and possibly safer than EtBr, though there is no data addressing its mutagenicity or toxicity in humans. SYBR Safe is a variant of SYBR Green that has been shown to have low enough levels of mutagenicity and toxicity to be deemed nonhazardous waste under U.S. Federal regulations. It has similar sensitivity levels to EtBr, but, like SYBR Green, is significantly more expensive.
Loading buffers are added with the DNA in order to visualize it and sediment it in the gel well. Negatively charged indicators keep track of the position of the DNA. Xylene cyanol and Bromophenol blue are typically used. They run at about 5000 bp and 300 bp respectively, but the precise position varies with percentage of the gel. Other less frequently used progress markers are Cresol Red and Orange G which run at about 125 bp and 50 bp.
After electrophoresis the gel is illuminated with an ultraviolet lamp (usually by placing it on a light box, while using protective gear to limit exposure to ultraviolet radiation) to view the DNA bands. The ethidium bromide fluoresces reddish-orange in the presence of DNA. The DNA band can also be cut out of the gel, and can then be dissolved to retrieve the purified DNA. The gel can then be photographed usually with a digital or polaroid camera. Although the stained nucleic acid fluoresces reddish-orange, images are usually shown in black and white
To separate DNA or RNA molecules by size
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.
When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, migrate towards the anode.
Agarose Concentration: By using gels with different concentrations of agarose, one can resolve different sizes of DNA fragments. Higher concentrations of agarose facilite separation of small DNAs, while low agarose concentrations allow resolution of larger DNAs.
Electrophoresis Buffer: Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity
Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels, as shown by all the images on this page. As described above, it can be incorporated into agarose gels, or added to samples of DNA before loading to enable visualization of the fragments within the gel. As might be expected, binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.
• An electrophoresis chamber and power supply
• Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis.
• Sample combs, around which molten agarose is poured to form sample wells in the gel.
Tris-borate-EDTA(TBE) ph8.2, 10x(100ml)
0.9M Tris HCl
4weigh 11.3 grams of Tris HCl
0.9M Boric acid
weigh 5.5 grams of boric acid
0.025 M EDTA
weigh 0.93 grams of EDTA
Dissolve the above chemicals in 100 ml of distilled water
Loading dye(6x)pH 7.6
10mM Tris HCl
0.03% Bromophenol blue
0.03%Xylene cyanol FF
Bromophenol blue and Xylene cyanol FF are for visual tracking of the DNA migration during electrophoresis
Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling.
Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromide-stained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light.
PREPARATION OF THE GEL
• To pour a gel,0.12 Grams of agarose powder is mixed with 5 ml of TBE (10x)buffer to this 45 Ml of distilled water is added and this is heated until completely melted.
• Then ethidium bromide is added to the gel (concentration10 microlitres) at this point to facilitate visualization of DNA after electrophoresis.
• After cooling the solution to about 60C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature
• After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber
• TBE buffer(1x) is poured inside the tank
LOADING OF THE SAMPLES
• About 15 microlitres of the sample is taken and to this 5 microlitres of the loading dye is added
• Both are mixed well and carefully loaded into the well without disturbing the gel
RUNNING OF THE GEL
• The lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards the positive electrode, which is usually colored red.
Observe the gel under a UV Transilluminator
Clear bands of DNA are observed.
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