Download DNA quantification

DNA concentration and purity determination
Why:
•Calculate how much to use in reaction or on gel
•Determine whether isolation was successful
•Determine whether DNA is clean enough to use.
DNA easily dissolves in aqueous solutions.
However, at high concentrations (10 mg/ml and
above), dissolved DNA is viscous. At lower
concentrations, one cannot detect the DNA by
sight or by noting the viscosity of the solution.
1. Concentration
• Concentration and quality of a sample of DNA or
RNA are measured with a UV
spectrophotometer.
• Since nitrogenous bases absorb UV light, the
more concentrated the DNA solution, the more
UV light it will absorb.
• A solution containing 50 µg per ml of double
strand DNA has an absorbancy (optical density)
of 1.0 at a wave length of 260 nm.
Unknown (ug/ml)/ Measured A260 = 50 (ug/ml)/ 1.0 A260
Unknown ug/ml = 50 ug/ml x Measured A260 x dilution factor
2. Quality
DNA quality measurement is based on the fact that OD
at 260 nm is almost twice that at 280 nm if the solution
contains pure DNA. If there is a contaminant, there is
some additional OD which decreases the OD ratio
between 260 and 280 nm.
• Clean DNA has a OD-260/OD280 approx~ 1.8-2.0
• If the measured value is smaller, it indicates a
contamination with protein or possibly phenol
• If the measured value is larger, it indicates a
contamination with RNA
3. a. Solution of pure double stranded DNA at 50ug/ml
has an absorbance of 1 at 260nm
b. Solution of pure single stranded DNA at 33ug/ml
has an absorbance of 1 at 260nm
c. Solution of pure RNA at 40ug/ml has an
absorbance of 1 at 260nm
Remember UV methods can be influenced by pH and
the ionic strength of the buffer.
Calculations
1.
OD 260nm=0.3, OD 280nm=0.6, what is the concentration of your
DNA and is it pure?
2.
You isolate DNA from bacteria and do a 1/10 dilution followed by a
second dilution of 1/100. You read the 1/100 dilution in the spec
and get OD 260nm=0.13 and OD 280nm= 0.08, what is the
concentration of your DNA and is it pure?
3.
You isolate DNA from human cells and take 0.12ul and add it to
0.2ml and then take 0.15ml and make it up to 860ul. You read this
solution in a spec at 26onm and get 0. 45, at 280nm you get 0.19,
what is the concentration of your DNA and is it pure?
4.
You isolate DNA from a virus with a single stranded DNA genome.
You take 233ul and make it up to 1034ul . The OD 260 is 0.3.
What is the concentration of DNA.
5.
You isolate RNA from a virus with a single stranded RNA genome.
You take 233ul and make it up to 1034ul . The OD 260 is 0.3.
What is the concentration of RNA