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Experiment 5

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Julia Garcia
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7 views

Experiment 5

Uploaded by

Julia Garcia
Copyright
© © All Rights Reserved
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Group Members : Costales Micah, Garcia Julia, Moises Jennifer

Date : March 11, 2023

Experiment 5
Determination of the Concentration and Purity of DNA
Guide Questions
1. Why did the experimenter dilute his DNA sample?
- “A thousand times less concentrated than the original sample”. The experimenter
diluted the sample because as he estimated the volume of his original sample,
he said that it is only 50 microliters which is not a very useful number, therefore,
to measure the sample he needed to dilute his original sample, especially since
he used spectrophotometer to measure the DNA sample, which only requires
small amount of the sample.
2. Why did he measure the absorbance at 260 nm? What chemical groups are
responsible for the absorption at this region?
- He measured the sample at 260 nm because for pure DNA samples, it is where
the maximum absorbance occurs and greater than that absorbance only absorbs
about half as much UV light compared to 260 nm.
- The chemical group responsible for absorption in this region is considered to be
nucleic acids as they strongly absorb UV light due to the resonance structure of
the purine and pyrimidine bases present in the DNA.
3. Why did he measure the absorbance at 280 nm? What chemical groups are
responsible for the absorption at this region?
- He measured the sample at 260 nm because for pure DNA samples, it is where
the maximum absorbance occurs and greater than that absorbance only absorbs
about half as much UV light compared to 260 nm.
- The chemical group responsible for absorption in this region is considered to be
nucleic acids as they strongly absorb UV light due to the resonance structure of
the purine and pyrimidine bases present in the DNA.
4. At what other wavelength do we measure absorbance to assess contamination of
DNA with organic impurities?
- Other wavelengths that is used to measure the absorbance to assess the
contamination of DNA with other organic impurities ranges from 230 nm to 320
nm.
5. The A260 reading was 0.014 when you diluted it 10 µL to 1 mL. Calculate the
concentration (in ng/µL) of the sample of DNA using this information. Show your
calculations.
- Formula: A260 x volume of the sample x dilution factor (1:100)
- Solution: 𝟏𝟎𝟎𝟎𝒖𝒍 ÷ 𝟏𝟎𝒖𝒍 = 𝟏𝟎𝟎𝒙 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏 𝒇𝒂𝒄𝒕𝒐𝒓
𝒏𝒈
𝟎. 𝟎𝟏𝟒 × 𝟓𝟎 𝒏𝒈/𝒖𝒍 × 𝟏𝟎𝟎𝒙 = 𝟕𝟎
𝒖𝒍
6. The UV spec registered absorbances of 0.009, 0.015, 0.012 at 230, 260, and 280
nm, respectively. What does this indicate about this sample of chromosomal DNA?
Show your calculations.
- The absorbance ratio of 260/280 is a good indicator of protein contamination, a
result of greater or equal to 1.8 indicates a pure DNA sample. While a result of
less than 1.8 from the absorbance ratio of 260/230 indicates contamination that
is probably caused by organic compounds or chaotropic agents which absorb at
230 nm.
- Formula: A260 / A280 ; A260 / A230
- Solution: (A260 / A280) = 0.015 / 0.012 = 1.25 ≤ 1.8
(A260 / A230) = 0.015 / 0.009 = 1.66 ≤ 1.8
- Conclusion: The result of 1.25 ≤ 1.8 from A260 / A280 ratio indicates that the
DNA sample is impure which indicates protein contamination as well as
contamination that probably caused by chaotropic agents (e.g., ethanol) which is
indicated based from the result of 1.66 ≤ 1.8 from A260 / A230 ratio.

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