DNA quantitation

OD (optical density) reading to quantitate purified plasmid DNA

Background

(adapted from Current Protocols in Molecular Biology, J. Wiley and

Sons)

Your purified plasmid DNA is ready to be transfected into mammalian cells; however, its concentration must be determined in order to calculate the correct amounts to use for downstream protocols, including transfection and any validation procedures prior to transfection. The concentration (and purity) of your DNA will be determined by spectrophotometry, with concentration typically expressed in units of µg/µl.

There are a number of common methods to quantitate the amount of DNA in a purified sample, and improvements are constantly being developed which increase the sensitivity of detection and require ever smaller volumes of sample.

As of now, the traditional, most convenient, and most commonly utilized method for determining the concentration and quantity of DNA in solution is to measure absorbance of a dilute solution of the DNA at 260 nm in a spectrophotometer, which is the approach we will use.

Both DNA and RNA absorb ultraviolet (UV) light at 260 nm, and A260 measurements are quantitative for relatively pure nucleic acid preparations in microgram quantities. (Your plasmid samples should be free of RNA due to the RNA-degrading enzyme “RNase”, used in buffer P1.) The Lambert-Beer law correlates absorbance of DNA (or RNA) to its concentration based on its extinction coefficient1 å :     A = å Cl

-where A is the absorbance at a specific wavelength (260nm for DNA and RNA);

-C is the concentration (in µg/ml);

-and l is the pathlength of the spectrophotometer cuvette (typically 1 cm).

The value for å for double stranded DNA (dsDNA) is 0.020 (µg/ml)-1 cm-1; rearranging the equation for concentration, and assuming a 1-cm-pathlength spectrophotometer cuvette, we have:

1 The molar absorptivity or molar extinction coefficient å of a chemical species at a given wavelength is a measure of how strongly the species absorbs light at that wavelength. It is an intrinsic property of the species; the actual absorbance of a sample is dependent on its thickness (l) and the concentration (C) of the species. In other words, å equals the absorbance per unit concentration (ug/ml or M/L) and per cm thickness.

C (in µg/ml) = A260 /0.020.

Using this equation, an A260 of 1.0 = 50 µ g/ml double-stranded DNA.

Calculating the concentration of your plasmid DNA:

The A260 value in the above equation consists of the instrument reading multiplied by the dilution factor of the sample. Small aliquots of each of your samples will be diluted by 1:00 for the spectrophotometer readings (see below), in order to bring your DNA concentrations into the linear range of detection for this type of assay. Also, the quartz cuvettes need at least 600 µl of liquid to register a reading. Your “raw” A260 instrument reading must therefore be multiplied by this dilution factor (100) to utilize the above equation.

For example, if your “raw” A260 = 0.2, then 0.2 x 100 = 20, which would be your

“real” A260 in this example.)

Continuing with this example, if:

A260 of 1 = 50 µg/ml, then A260 of 20 = 20 x 50 µg/ml, = 1000 µg/ml (or 1 mg/ml or 1 µg/µl). Therefore, a spectrophotometer reading of 0.2 would mean that you had a DNA concentration of 1000 µg/ml.

As a shortcut: we will rearrange the extinction coefficient and convert from ml to µl (convince yourself that this is correct):

Refer back to the equation: C (in µg/ml) = A260 /0.020

or:   • C (in µg/ml) = A260 x 1/0.020

  • C (in µg/ml) = A260 x 50
  • C (in µg/µ l) = A260 x 50 x 1/1000
  • C (in µg/µ l) = A260 x 0.05

Rewriting the last equation to include our dilution factor, you have:

C (µg/µl) = Instrument Reading260 x 100 x 0.05

or

C (μg/μl) = Instrument Reading260 x 5

 

So, simply multiply your “raw” A260 reading by 5 to obtain your concentration in units of μg/μl DNA. For example, an instrument reading of 0.1 means you have 0.5 μg/μl of DNA.

Most proteins in solution have an absorption maximum at 280 nm due to the presence of tryptophan, tyrosine, and phenylalanine residues. (However, the molar absorptivity of each individual protein varies greatly, depending on its content of these amino acids.) Therefore, the absorbance at 280 nm is also read to determine the amount of contaminating protein in the DNA sample. The A260/A280 ratio gives a reliable indication of the “cleanliness” of the DNA preparation. Ideally this number should be between 1.8 and 2.0 (the value of a protein-free DNA prep). (The reason that a pure, protein-free DNA preparation would have any absorption at 280 nm is because, whereas the purine and pyrimidine rings of nucleic acids have absorption maxima near 260 nm, there is still considerable absorption extending as high as 280 nm.)

A260/280 ratios that are less than 1.8 indicate a degree of protein contamination of the DNA prep (although not necessarily harmful for our purposes); ratios that are greater than ~2.0 indicate contamination from RNA (although that is not expected to be an issue with our preps, due to RNase in the initial resuspension buffer)

Protocol: OD (optical density) reading to quantitate purified plasmid DNA

Reagents:

  • Purified plasmid DNA
  • Quartz cuvette
  • TE buffer to “blank” the machine

Lab Instrumentation:

Insert image

UV/Vis Spectrophotometer

  1. Turn on the spectrophotometer2 (switch on back left). Lift the front screen. Choose “nucleic acid” from the main menu by pressing the arrow on the keypad. Select number 1: 260/280 ratio. The UV lamp will begin to warm
  2. It takes approximately 10 minutes until the lamp is ready to measure your sample.
  3. Dilute 6 µl of each plasmid DNA sample into 600 µl of TE buffer in a new microcentrifuge tube. Vortex and bring samples to the spectrophotometer.
  4. To “blank” the machine before the class readings (done once): we will pipet 600 µl of TE buffer into the quartz cuvette and place in the cuvette holder. Then press “Blank “. The machine will calibrate on this sample, which will effectively subtract the background reading.
  5. Remove the buffer from the cuvette by pouring into the nearby waste beaker. Pipette in your first sample to be read.
  6. Press “Read”.
  7. Record the absorbance readings and the A260/A280 ratio. Remove the sample, rinse the cuvette a few times with dH20 from a squeeze bottle, shake to dry and proceed to the next sample.
  8. Calculate the concentrations of DNA (in µg/µl) in each of your plasmid samples as indicated in the background section (above); record the concentrations and the 260/280 ratios.

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google+ photo

You are commenting using your Google+ account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s