CHAPTER 1 (QTFCATION OF NA)

Question 1

how to determine the conc. of NA

Answer 1

using spectrophotometer

Question 2

explain the usage of spectrophotometer

Answer 2

Sample absorbance is determined on the
spectrophotometer at 260nm as NA (DNA,
RNA, nucleotides) absorb light at 260nm

Question 3

One optical density or absorbance unit at
260 nm is equal to

Answer 3

50 µg/mL for dsDNA
40 µg/mL for RNA.
33 µg/mL for ssDNA

Question 4

the formula of DNA concentration (µg/ml)

Answer 4

(OD 260) x (dilution factor) x (50 µg DNA/ml)

Question 5

the formula of RNA concentration (µg/ml)

Answer 5

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

Question 6

A volume of 1 ul of a dsDNA
sample was diluted in 100ul and
the OD of the sample was
determined as 1.6.

Calculate the concentration of the
sample above. If the sample was
RNA instead, what would the
concentration be?

Answer 6

DF = 100X
OD260 = 1.6
DNA/ml = 50

100x1.6x50 =8,000 µg/ml

Question 7

how to determine purity of NA

Answer 7

using spectrophotometer by measuring its wavelength at both 260nm and 280nm

Question 8

the formula to calculate purity of NA

Answer 8

• Divide the OD at 260nm by the OD at 280nm to get the OD260/OD280 ratio
• The OD260/OD280 ratio is a measure of NA purity

Question 9

whats the ideal ratio for DNA, RNA and Protein?

Answer 9

DNA: 1.8
RNA: 2.0
Protein: 0.6

Question 10

what does it mean if ratio is less than ideal

Answer 10

protein contamination

Question 11

what does it mean if ratio is higher than ideal

Answer 11

for DNA, it could be related to DNA degradation

for both, it can mean lower conc. of NA in sample