NAME

UMAR AZIZ

ASSIGNMENT

HOW TO PERFORM DNA QUANTIFICATION

POULTRY GENOMICS
DNA Quantification
DNA quantification and RNA quantification, generally referred to as nucleic acid quantification, is
commonly performed to determine the average concentration of DNA or RNA in a sample prior to
proceeding with downstream experiments. Sample purity is also an important consideration to
accurately calculate the amount of DNA or RNA in a sample. There are two optical technologies
commonly used quantify nucleic acids: UV-Vis measurement and fluorescence measurement. Choosing
the right technology for your samples, workflow and throughput results in accurate RNA or DNA
quantification and can save significant time and money by helping to prevent downstream experimental
failures. In current era of NGS technologies, most of sequencing manufacturer recommends to have
dsDNA-specific fluorometric quantitation methods.  It is important to choose fluorometric methods of
DNA quantitation that are specific to double-stranded DNA (dsDNA), since single-stranded DNA
(ssDNA) is not a suitable substrate for most library preparation technologies.

QUANTIFICATION OF DNA, RNA

 Concentration of dsDNA= 50 µg/ml x Dilution factor x (Absorbance at 260 Nm-

absorbance at 320 Nm)

 Concentration of ssDNA = 33 µg/ml x Dilution factor x (Absorbance at 260 Nm-

absorbance at 320 Nm)

 Concentration of RNA = 40 µg/ml x Dilution factor x (Absorbance at 260 Nm-

absorbance at 320 Nm)

Material:
 DNA/RNA/Protein sample

 Buffer

Method:
1. Start on spectrophotometer and adjust it to wavelength 260 nM.

2. Auto-zero spectrum with blank of TE buffer for DNA/RNA or PBS buffer Protein sample.

3. Withdraw buffer and rinse cuvette once with distilled water.

4. Fill cuvette with max volume with DNA/RNA/Protein sample.

5. Run absorbance spectrum in range of 230 to 330 Nm.

6. Run Peak value and find point with maximum absorbance in range of scan and note it

7. Further, take absorbance at 230, 260 and 280 Nm and note it.

8. Check quality of sample by taking ratio of A230/260/280, A260/280 and A260/230.

9. Calculate quantity of sample as per equation and note it.

Direct Calculation
Percent methylation compared to the control DNA

1. Average all control, sample, and blank absorbance replicates.

2. Subtract the average blank value from the average control and sample values.

3. Divide the background subtracted average sample by the background subtracted average
normal sample.

4. Multiply by 100.

5. Sample calculation:
Average Blank Absorbance = (A450) = 0.090
Average Absorbance for 60 ng control DNA = 0.785
Average Absorbance for 60 ng sample DNA = 0.654

   0.654 - 0.090 x 100 = 81.15%

   0.785 - 0.090

The sample DNA has a global methylation level that is 81.15% of the control DNA.

Calculation from a standard curve

Percent methylation relative to DNA used to generate the standard curve

1. Determine the concentration of DNA in the sample(s) by A260 or fluorimetric means.

2. Create a standard curve by plotting background subtracted A450 versus control DNA
(see example)
3. For each background subtracted sample reading, calculate the % methylation as below:
Methyl DNA (ng) = [A450 background subtracted – intercept] / slope
                                                          ng in sample

Note: in many or most cases the intercept will be zero.