DNA Quantification and Measurement of Purity Flashcards
dsDNA
double stranded deoxyribonucleic acids
dsDNA quantitfication is necessary for efficient ______ and ______.
HLA genotyping and sample conservation
Two values that are determined by dsDNA quantification?
DNA concentration and DNA purity
DNA Quantification methods
UV absorbance spectroscopy (traditional method for tissue), fluorometric methods, gel electrophoresis, RTPCR
Beer-Lambert equation
Absorbance of DNA in solution
A=ebC
e=molar extinction coefficient
b= path length
C= concentration
What is the extinction coefficient for dsDNA, single stranded DNA (ssDNA) and RNA?
dsDNA =50
ssDNA =33
RNA =40
When the molar extinction coefficient and reader path legnth are ___________, abosrobance is proportional to the concentration of DNA.
constant
What is the path length for a standard cuvette reader?
1 centimeter
Beer-Lambert equation is only linear for absorbance between _______ and _______.
0.1 - 1.0
Spectrophotometric methods that measure UV absorbance should be avoided because the presence of what contaminants can contribute to overall UV absorbance?
RNA, ssDNA, or oligonucleotides
PicoGreen
ultra-sensitive fluorescent dye that undergoes a dramatic fluorescence enhancement upon binding to dsDNA that can be measured using a fluorometer. Used for quantification of DNA amplification products.
Linear detection range extends four orders of magnitude in DNA concentration - from 1 ng/mL to 1000 ng/mL with a single dye concentration
developed to minimize the contribution of RNA and ssDNA
How is linearity maintained in PicoGreen method?
salts, urea, ethanol, chloroform, detergents, proteins, agarose
What equation must be used to quantify the DNA sample when using a spectrophotometer?
DNA in ug/uL = (50 x dilution factor x absorbance at 260nm) / 1000
