DNA concentration and purity determination

1. 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.

2. 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

3. 2. QualityDNA 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

5. 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.

6. Calculations • OD 260nm=0.3, OD 280nm=0.6, what is the concentration of your DNA and is it pure? • 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? • 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? • 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. • 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