QUALITATIVE AND QUANTITATIVE ANALYSIS OF DNA BY SPECTROPHOTOMETRY

 

PROCEDURE
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

For example:

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

Cycles                                                  35

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.

Ethanol precipitation-
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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how the DNA is diluted 100 times?

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