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QUALITATIVE ANALYSIS OF PLASMID DNA BY AGAROSE GEL ELECTROPHORESIS Agarose gel electrophoresis is a technique most commonly used for

separation based on molecular size in the presence of electric current. Ethidium bromide, a fluorescent dye that is used to detect DNA in agarose, reduces the electrophoretic mobility of linear DNA by about 15%. Larger molecules of linear double stranded DNA migrates more slowly because of their greater frictional drag and pass through the pores of the gel less efficiently than smaller molecules. Super helical (form I), nicked circular (form- II) and linear (form-III) DNA of the same molecular weight migrate through agarose gels at different rates. The relative mobilitys of the three forms depend primarily on the agarose concentration in the gel, but the strength of applied current, the ionic strength of the buffer and the density of the super helical twists in the form I DNA also influence them. TAE is the most commonly used buffer. Agarose gels are usually run in horizontal configuration. If electric field is applied across the gel, DNA, which is negatively charged at neutral pH, migrates towards the anode. The samples get separated based on their molecular size and charge. Hence by using a marker DNA, the size can be found out . Agarose gels have a lower resolving power than the polyacrylamide gels but have a greater range of separation. PROTOCOL:
STEP I - PREPARATION OF THE GEL

1. The edges of a clean, dry glass plate were sealed with tape, so as to form a mold. The combs were positioned 0.5 - 0.1 mm above the plate, so that the complete well is formed when the agarose is added. Two combs with six wells each were placed so as to get 12 wells in a single gel. 2. Sufficient electrophoresis buffer (1X TAE) was prepared - to fill the electrophoresis tank and to prepare the gel. 3. 0.8% agarose gel was prepared by dissolving 0.4g of agarose in 50ml of 1x TE buffer by heating at a temperature above 600C. The solution was cooled to 60oC and Ethidium

Bromide (From a stock solution of 10mg/ml in water) was added to get a final concentration of 0.5g/ml. 4. The gel was poured in gel casting unit with combs. 5. After the gel was completely set, the combs and tapes were carefully removed and the gel was mounted in the electrophoresis tank. 6. Enough electrophoresis buffers, to cover the gel to a depth of about 1mm was poured in the electrophoresis tank.

STEP-II

LOADING OF SAMPLES:

10l of DNA samples were taken in a separate eppendorf tubes and 1.5l of gel loading buffer was added to each tube and mixed well. (The gel-loading buffer is available as a stock of 6X concentration. Thus based on the volume of DNA sample, loaded dye is added to give a final concentration of 1X.)

Gel loading buffer serves 3 purposes: It increases the density of sample, ensuring that the DNA drops evenly into well. They add color to samples, thereby simplifying the loading process. They contain dyes, which move towards the anode at predictable rates in the electric field. Bromophenol blue and xylene cyanol are the two dyes present in the loading dye. Bromophenol blue migrates through agarose gel approximately 2.2 fold faster than xylene cyanol independent of agarose concentration. Bromophenol blue

migrates through agarose gels approximately at the same rate as linear double stranded DNA of 300 bp in length and xylene cyanol migrates at approximately the same rate as 4kb of linear double stranded DNA.

LOADING PATTERN: 1. With the help of micropipette, the samples were loaded into appropriate wells. 2. The lid of gel tank was closed and electric leads were attached, so that the DNA will migrate towards the anode. A voltage of 50-100V/cm was applied. The gel was run until

bromophenol blue and xylene cyanol FF have migrated the appropriate distance through gel. 3. After electrophoresis for 1:30 to 2:00 hours, the electric current was turned off and leads were removed. transilluminator. The lid of tank was opened and the gel was observed under U.V

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