DNA Quantification

Question 1

Why is it necessary to measure DNA

Answer 1

Concentration of DNA Is NB as if too low or too high, it will affect PCR
• Checks for purity and possible contamination
• It is essential to have a reliable measurement of DNA concentration because DNA is vital in several applications in molecular biology.
• Spectrophotometry and fluorometry are often utilised to quantify genomic and plasmid DNA concentrations.
• Spectrophotometry can measure microgram quantities of pure DNA samples (i.e.,
DNA that is not contaminated by proteins, phenol, agarose, or RNA).

Question 2

DNA absorbance

Answer 2

DNA concentration is determined by quantifying the absorbance
at 260 nm (A260) in a spectrophotometer (Nanodrop).
• The reading must be between 0.1 and 1.0 to obtain accuracy.
DNA quantification
• An absorbance of 1 unit at 260 nm corresponds to 50 ug genomic
DNA per ml (A260 =1 for 50 ug/ml; based on a standard 1 cm path
length.

Question 3

DNA and pH

Answer 3

-All genomic measurements must be ade at neutral pH
-Higher or lower pH can result in mis-interpretation by spectrophotometer
-Samples must have a low-salted buffer with a neutral pH

Question 4

DNA and RNA purification

Answer 4

-RNA will often be co-purified with genomic DNA
-RNA may inhibit some downstream applications but it will not inhibit PCR

Question 5

RNA detection

Answer 5

RNA contamination can sometimes be detected by agarose gel analysis with ethdium bromide staining, although not effectively
RNA bands appear faint and smeary and are only detected in amounts > 25-30ng

Question 6

DNA sequencing

Answer 6

DNA sequencing determines the precise order of nucleotides within a DNA
-Different methods are used to determine the order of the bases
-This info is useful to various fields of biology and other sciences, medicine, forensics, biotechnology, virology, and other areas of study

Question 7

Colors of bases

Answer 7

Adenine-Green
G-black
C-blue
T-red