ELUTION OF SEPARATED BANDS OF VECTOR AND GENE INSERT FROM AGAROSE GEL PRINCIPLE: There are several methods

available for purification of DNA molecules from polyacrylamide and agarose gels. Low temperature melting agarose gels can be used to isolate DNA fragments, as its melting point is 65C. This temperature will not cause harm to the DNA but it is capable of melting the agarose. In this agarose, hydroxyl-ethyl groups have been introduced into the polysaccharide chains. This causes agarose to gel at approximately 30C and melt at around 65C well below the melting temperature of the double stranded DNA. This property is exploited to make the recovery much easier. PROTOCOL: 1. DNA was digested with desired restriction enzymes and fractionated electrophoretically through an agarose gel that contains ethidium bromide of 0.5g/ml. 2. After electrophoresis, the bands were located under ultraviolet fluorescence. An incision was made in the gel ahead of the desired band (towards positive electrode. A rectangle piece of agarose was cut). 3. The agarose along with the band was taken out and transferred into fresh Eppendorf tubes and crushed well 4. Then 2.5M Ammonium acetate (350l) was added to the crushed well until the agarose completely dissolves in the tube. 5. Centrifuge at 12,000 rpm for 5min at 40C. 6. Take supernatant in fresh eppendorf tubes and add 2.5 volumes of absolute ethanol and store at 200C for overnight. 7. Centrifuge at 12,000rpm for 20 min 40C to get DNA pellet. 8. The pellet was washed with 70% ice cold ethanol. 9. This was centrifuged at 12,000rpm for 10 min at 40C 10. The pellet was dried in speed vacuum desiccators for 5 to 7 min and dissolved in 50l of 1X TE

ESTIMATING THE CONCENTRATION OF DNA IN SMALL VOLUME USING SPOT TEST: It is often desirable to determine the concentration of a DNA fragment preparation. Two methods are widely used for estimating the concentration of the amount of nucleic acid in preparation. Spectrophotometric measurement of UV radiation absorbed by the nucleotide bases is a simple and accurate. If the sample contains significant quantities of impurities the amount of nucleic acid can be estimated from the intensity of the fluorescence emitted by Ethidium bromide. However spectrophotometry is often not a practical due to the small volume of the

fragment preparation .so the spot test is frequently used method for estimating the concentration of nucleic acid. PROTOCOL: 1. Standard solutions of known DNA concentrations were prepared in the range of 0-50 ng/l in TE or distilled water. 2. On a piece of parafilm, 2.5l of 5ng/l solution of ethidium bromide was arranged and a standard curve was prepared by mixing 2l of standard DNA solution each. 0,1,5,15,30 and 50 ng/l standards were used. 3. The samples of DNA fragment preparation (2l) were mixed each with 2.5l of ethidium Bromide on a parafilm. 4. The parafilm was then placed on a UV transilluminator and visualized under UV light.
5. The fluorescence of the DNA fragment preparation was compared with the standard

curve in order to estimate its concentration.