2
Concentration, yield and purity of DNA
Spectrophotometric analysis and agarose gel electrophoresis will reveal the concentration and
the purity of the genomic DNA. Use Elution Buffer (ET) to dilute samples and to calibrate the
spectrophotometer, measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz
micro cuvette.
Absorbance readings at 260 nm should fall between 0.1 and 1.0. The 320 nm absorbance is used
to correct for background absorbance. An absorbance of 1.0 at 260 nm corresponds to
approximately 50 µg/ml of DNA. The A
260
-
A
320
/A
280
-A
320
ratio should be 1.6-1.9. Purity is
determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. DNA
purified by Insta NX® Fungal DNA Purification Kit is free of protein and other contaminants that
can inhibit PCR or other enzymatic reactions.
Concentration of DNA sample (µg/ml) = 50 x A
260
x dilution factor.
Storage
Store the Insta NX® Fungal DNA Purification Kit between 15-25°C except certain components as
specified on each labels. Under recommended condition kit is stable for 4 months.
Materials needed but not provided
Tabletop Microcentrifuge (with rotor for 2.0 ml tubes) capable of upto
≈
14,000 rpm
(LA1001)
65°C water bath or heating block
Vortex Mixer
Insta NX® (LA1056)
Molecular Biology Grade Water (ML024)
Micropipettes (LA617, LA613) and Tips (LA974)
General Preparation Instructions
IMPORTANT: Please go through the instruction manual before starting the experiment.
1. Preheat a water bath or heating block to 65°C.
RNase A enzyme treatment
RNase A is a type of RNase that is commonly used in research. RNase A (e.g. bovine pancreatic
ribonuclease A) is one of the sturdiest enzymes in common laboratory usage. It cleaves 3' end
of unpaired C and U residues.
Unit Definition for RNase A
One unit of the enzyme causes an increase in absorbance of 1.0 at 260 nm when yeast RNA is
hydrolyzed at 37°C and pH 5.0. Fifty units are approximately equivalent to 1 Kunitz unit. It is
completely free of DNases and proteases. The specific activity is 90 U/mg.
The product as supplied is stable at room temperature (15-25°C).
