Spectrophotometric Analysis
PRINCIPLE: The easiest way of determinig DNA concentration is through spectrophotometric analysis.Since nitrogenous bases absorb UV light,the more concentrated the DNA solution, the more UV light it will absorb. The concentration of pure double stranded DNA
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with an A260of 1.0 is 50mg/ml.Unknown=50mg/ml*measured A260*dilution factor(mg/ml)
PROCEDURE:
1. The DNA samples were all pooled in together.
2. Approximately, 150-200ul of total volume was obtained.
3. 10ul of DNA sample was taken from this pool and 1490ul of autoclaved distilled water was added to it, there by resulting in the test(unknown) volume of 1500ul
4. Blank solution was 1500ul of distilled water.
5. Spectrophotometer was set according to the instructions given and the O.D(Optical Density) values were recorded.
6. The concentration was unknown(test) samples was calculated using the standard formula.
CALCULATIONS:
Unknown=50ug/ml*measured A260*dilution factor
=50*0.345*150
=2587.5ug/ml
=2.5mg/ml
INFERENCE: Quantifications of the unknown(test) DNA sample was done, after obtaining the absorbance value A260. Due to some experimental error, the unknown DNA samples' concentration calculated came out to be too high. This can be supported by the fact that the gel showed a band of RNA contamination.
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