QUALITATIVE AND QUANTITATIVE ANALYSIS OF DNA BY SPECTROPHOTOMETRY
Take 3ml of SSC buffer in the cuvette as blank .
Add 50μl of isolated DNA and make the volume up to 3ml by adding 2950µl ssc buffer.
Place the cuvettes in the spectrophotometer and measure the absorbance at 260nm and 280nm against blank .
The purity if the nucleic acids is determined by calculating the ratio of absorbance at both 260 and 280nm by using the following formula. O.D 260 nm/ O.D 280 nm. Concentration of DNA (µg/ml)=O.D.260X50Xdilution factor
Sample No O.D.260 O.D.280 O.D.260 / O.D.280
Sample 1 0.126 0.233 0.233/0.123=1.849
Sample2 0.118 0.226 0.266/0.118=1.915
Although O.D.260 / O.D.280 ratio of both the samples 1 and 2 is between 1.8 and 2.0 but sample A DNA Is better and pure because the ratio is close to 1.8.
While taking absorbance, the sample is diluted 100times, i.e. dilution factor is 100 times Conc. of DNA of sample1 = 0.233X50X100=1165µg/ml = 1.165µg/ml.
Quantification of plasmid DNA:-
Isolated DNA was quantified by measuring the absorbance at 260 and at 280 nm. The ratio of absorbance 260/280 was used to determine the quality of the isolated DNA .The concentration was calculated using the following formula
Concentration of the DNA = OD260 X 50 X dilution factor = mg of DNA /ml
Quantified DNA was diluted to 150 ng/ml and used for polymerase chain
Polymerase chain reaction-
Polymerase chain reaction was performed using primers that were designed from the pUC19 vector sequence (Table). Primers were commercially obtained from Sigma–Aldrich (Sigma-Aldrich Inc. St. Louis, Missouri 63178, USA). All the primers designed were 20 base pairs or more in length with GC content in the range of 40-80%. Tm was calculated according to the formula Tm(C) = 2(A+T) + 4(G+C) -5. The annealing temperature for each pair of PCR primers was optimized experimentally.
PCR amplification was carried out with following reaction parameters
dNTPs 200 µM
PCR reaction buffer 1X
Magnesium chloride 1.5 -2.5 mM
Primer (forward) 50 pmoles
Primer (reverse) 10 pmoles each
Templet DNA 150 ng
Taq DNA polymerase 1U
The final reaction volume was 50 µl.
The cycling conditions used:
Initial denaturation 94oC, 2 min
Denaturation 94oC, 45 sec
Annealing 54-62oC, 30sec
Elongation 72oC, 45 sec
Final elongation 72oC, 10 min
Agarose gel electrophoresis:
0.8 to1.5% agarose gels were prepared by dissolving the required quantity of agarose in 1X Tris-Acetate-EDTA (40mM tris-acetate. 1mM EDTA) electrophoresis buffer by heating in a microwave oven, followed by addition of ethidium bromide to a final concentration of 0.25 ug/ml. The agarose solution was poured into a gel tray containing a comb, allowed to cool and solidify, and then placed in electrophoresis tank and submerged in 1X TAE buffer. DNA samples were mixed with 6X gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol 40% w/v sucrose). Samples were loaded on the gel along with DNA size standards. Horizontal electrophoresis was carried out at approximately 80-100V. The gel was photographed under UV light using the gel documentation system.
Ice cold ethanol (100%) was added in a ratio of 1:5 (500 ul polled reaction mix and 2.5ml of ice cold ethanol). The mix was mixed properly and left at -80degC for 1 hour. After the incubation the DNA was precipitated by spinning at 12000rpm for 10min. The pellet is washed in cold 70% ethanol then after a further centrifugation step the ethanol is removed, and the nucleic acid pellet is allowed to dry before being resuspended in nuclease free water.
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