Sesperes, John Kenneth

Tan, Hannah Michaela

Tapia, Paul Adrian

Tarriela, Mark Khim

Viray, Danielle

Grp.5 - HUB42

• The concentration of an RNA or DNA sample can be checked by the use of UV spectrophotometry.

• Both RNA and DNA absorb UV light very efficiently making it possible to detect and quantify either at concentrations as low as 2.5 ng/µl.

• The nitrogenous bases in nucleotides have an absorption maximum at about 260 nm.

• Using a 1-cm light path, the extinction coefficient for nucleotides at this wavelength is 20.

• Based on this extinction coefficient, the absorbance at 260 nm in a 1-cm quartz cuvette of a 50µg/ml solution of double stranded DNA or a 40µg/ml solution of single stranded RNA is equal to 1.

DNA concentration (µg/ml) = (OD 260) x (dilution factor) x (50 µg DNA/ml)/(1 OD260 unit)

RNA concentration (µg/ml) = (OD 260) x (dilution factor) x (40 µg RNA/ml)/(1 OD260 unit)

• The absorbance of a DNA sample at 280 nm gives an estimate of the protein contamination of the sample.

• Purification of intact nucleic acid from samples is required for many molecular biology applications.

(e.g. PCR)• The assessment of the purity of a nucleic acid sample is often performed by a procedure commonly referred to as the A260/A280 ratio.

by Warburg and Christian originally this was used to measure protein purity in the presence of nucleic acid contamination

• Pure DNA: 1.80 – 1.90

• Pure RNA: 2.0

• The basis of this test rests on the Beer-Lambert Law

OD = eCb

Wherein:

OD = optical density

e = extinction coefficient

C = concentration of the sample

b = optical path length

e = OD/C

WavelengthWavelength AbsorbanceAbsorbance

220220 0.0400.040

240240 0.0000.000

260260 (-) 0.004(-) 0.004

280280 0.0320.032

300300 0.020 0.020

Table 1.1 Absorbance of DNA sample extracted from E.coli at various wavelengths

1st Sample: A260 / A 280 = 0.125

2nd Sample:

WavelengthWavelength AbsorbanceAbsorbance

220220 (-) 0.039 (-) 0.039

240240 (-) 0.033 (-) 0.033

260260 0.011 0.011

280280 (-) 0.025(-) 0.025

300300 (-) 0.077 (-) 0.077

Table 1.2 Absorbance of DNA sample extracted from E.coli at various wavelengths

A260 / A 280 = 0.44

0

0.02

0.04

0.06

0.08

0.1

0.12

220 240 260 280 300

2nd sample

1st sample

Figure 1.1 Absorbance profiles of DNA sample extracted from E.coli at various wavelengths

ONION DNA ABSORBANCE

 Readin

g260 nm

280 nm

RATIO

TRIAL 1

1 0.000 -0.023 0.000

2 -0.005 0.570 -0.009

3 -0.157 -0.126 1.246

4 -0.043 -0.022 1.955

TRIAL 2

1 -0.028 0.027 -1.037

2 -0.022 -0.011 2.000

3 -0.064 -0.030 2.133

4 -0.046 0.015 -3.067

Table 1.3 Absorbance of DNA sample extracted from Onion at 260 and 280 nm

• It is important to note that the A260/A280 ratio isonly an indication of purity rather than a precise answer.

• Several factors may influence A260/A280 ratios.

1.Measurements at 260 nm are near the nucleicacid absorbance spectrum peak, while those at280 nm are located in a portion of the spectrumwith a steep slope.

2.Sample concentration can also affect the results, as dilute samples will have very little difference between the absorbance at 260 nm and that at 280 nm.

3.The type(s) of protein present in a DNA/protein mixture can also affect the A260/A280 ratio determination.