Measurement Of DNA Concentration By Spectrometry
SP, July 2004
Using Ultrospec 2000 spectrophotometer
DNA absorbs light of certain wavelength so that light of 260 nm passing 1 cm through DNA at 50 ug/ml concentration (in water) has an absorbance of 1.0.
The spectometer is set to use light of 260 nm. A negative control or blank cuvette (tube) is filled with water or just the buffer solution (such as TE; usually water) that the DNA is in. As the tube has no DNA, it should give an OD reading of 0. The machine however may show the reading to be something else. The machine is therefore 'zeroed' to set the reference at 0.
The sample to be analysed is diluted, usually 50-500 fold (usually 200-300 fold) in water (or buffer). The solution is puured into a similar-sized cuvette as the one used for blank/negative/reference and its OD measured.
The cuvettes used in Ultrospec 2000 have a 'path length' of 1 cm.
Multiplying OD (260 nm absorbance) with 50 ug/ml will give DNA concentration (inside the cuvette) and a further multiplication with the dilution factor (50-500) will give DNA concentration in the sample.
example: A sample has been diluted 100 fold in water (i.e. 10ul sample in 990ul water). After analyzing the cuvette in the spectrophotometer machine, you get an OD reading of 0.008 on 260nm absorbance setting. Multiplying this OD number by 50 ug/ml gives a concentration inside the cuvette of 0.4 ug/ml. A further multiplication of 100 will give a concentration in the sample of 40 ug/ml, which is equivalent to 40 ng/ul.
Why is a volume measured in ug/ml numerically equivalent to a volume expressed in units of ng/ul? Follow the units conversion using dimensional analysis: X ug/ml * 1ml/1000ul * 1000ng/1ug = X ng/ul (because the 1000's cancel out as well as the units "ml" and "ug").
— Using 1 ml UV (can pass through) disposable cuvettes (in drawer under the spec). Cuvettes with smaller volumes but same path length are also available for use, especially if the sample amount is small. They are reusable and should be rinsed with water before and after use.
Note that cuvettes are asymmetric, meaning they should be placed in the cell inside the spec in a certain way. Usually the 1 cm dimension should be along the L-R axis.
— Turn on the spec, the power button is at the back on the right side. It takes a minute or so to get warmed up.
— Prepare 1ml of 50-500x dilution of sample (usually 3-4 ul plasmid DNA or 5-10 ul purified PCR product in 1ml water) in the disposable UV cuvettes. Make sure the DNA is mixed well and that there are no bubbles inside, or liquid droplets on the outside walls. Also prepare a blank using just 1 ml water.
— Press (WAVE) button and use the (1), (2), etc. digit keys to key in 260 and then press (ENTER)
— Put the blank cuvette in and close the cover.
— Press (SET REF) to zero the reading to 0.
— Remove cuvette and place your sample cuvette. Close cover. The OD reading will change from 0 to a different value. record it. repeat for all samples.
— Calculate DNA concentration as described earlier.
— Useful when multiple samples; set all sample and blank cuvettes inside (blank cuvette first; in cell no. 1, blue colored)
— (MODE) key, press till 'DNA' on display; (ENTER)
— Display shows 'background? y/n.' Use < or > key and (ENTER)
Sets background correction; useful when using high concentration buffers or low concentration samples.
— (SET REF) key
— (RUN) key. After a few seconds the display will show DNA concentration for the first sample (2nd cuvette, in 2nd cell) in ug/ml. Note that that is the concentration of the diluted sample in the cuvette.
— Press (RUN) key again to move to the next sample.