Chapter 4 Nucleic Acid Extraction Methods

DNA Quantity/Quality

Calculate the DNA concentration in µg/mL from the following information:

a. Absorbance reading at 260 nm from a 1:100 dilution = 0.307

b. Absorbance reading at 260 nm from a 1:50 dilution = 0.307

c. Absorbance reading at 260 nm from a 1:100 dilution = 0.172

d. Absorbance reading at 260 nm from a 1:100 dilution = 0.088

Nucleic acids absorb light at 260 nm through adenine residues.

Using Beer-Lambert Law, concentrationcan be determined from the absorptivity constants. 

50 for double-stranded DNA
40 for RNA

The relationship of concentration to absorbance is expressed as

A = €bc

A = absorbance,
€= molar absorptivity (L/mol-cm)
b = path length (cm)
c = concentration (mg/L).

a. Absorbance reading at 260 nm from      1:100 dilution = 0.307

0.307x50 ug/mL = 15.35 ug/mL   15.35 ug/mLx100 = 1535 ug/mL


b. Absorbance reading at 260 nm from a 1:50 dilution = 0.307

0.307x50 ug/mL = 15.35 ug/mL   15.35 ug/mLx50 = 767.5 ug/mL


c. Absorbance reading at 260 nm from a 1:100 dilution = 0.172

0.172x50 ug/mL = 8.60 ug/mL   8.60 ug/mLx100 = 860 ug/mL


d. Absorbance reading at 260 nm from a 1:100 dilution = 0.088

0.088x50 ug/mL = 4.40 ug/mL
4.40 ug/mLx100 = 440 ug/mL


If the volume of the above DNA solutions was 0.5 mL, calculate the yield for

(a) – (d).

Three DNA preparations have the above following A260 and A280 readings:

Each sample based on the A260/A280 ratio, is each preparation suitable for further use?

If not, what is contaminating the DNA?

After agarose gel electrophoresis, a 0.5 µg aliquot ofDNA isolated from a bacterial culture produced only a faint smear at the bottom of the gel lane.

Is this anacceptable DNA sample?

Contrast the measurement of DNA concentration by spectrophotometry with analysis by fluorometry with regard to staining requirements and accuracy.

RNA Quantity/Quality

Calculate the RNA concentration in µg/mL from the following information:

a. Absorbance reading at 260 nm from a 1:100 dilution = 0.307.

b. Absorbance reading at 260 nm from a 1:50 dilution = 0.307.

c. Absorbance reading at 260 nm from a 1:100 dilution = 0.172.

d. Absorbance reading at 260 nm from a 1:100 dilution = 0.088.

If the volume of the above RNA solutions was 0.5 mL,calculate the yield for (a)–(d).

An RNA preparation has the following absorbance readings:

A260= 0.208
A280= 0.096

Is this RNA preparation satisfactory for use?

A blood sample was held at room temperature for 5 days before being processed for RNA isolation. Will this sample likely yield optimal RNA?

Name three factors that will affect yield of RNA froma paraffin-embedded tissue sample.