GFP Project: DNA Quantification

This is the nanodrop, a machine for quantifying DNA.

So now that we have grown up and extracted our plasmid DNA, the next question is how much do we have?  And how the heck do you measure the concentration and purity (amount of unwanted protein to amount of DNA) of nanograms of DNA dissolved in microliters of volume?

The answer is that we do these things with spectrometry magic.  It turns out that proteins (which are all made of amino acids) tend to absorb light at 280nm, while nucleic acids tend to absorb light at 260nm.  The concentration can be determined by the absorbence at 260nm (A260), and it turns out that “pure” nucleic acid samples have an A260/A280 absorbence ratio of 1.8 or higher.

This can be done with a spectrophotometer and nice quartz cuvettes, but this means you need enough liquid to fill the bottom of the cuvette, so you have to do a dilution.  This means more math to figure out the actual concentration, and it means that you have to do the figuring of the ratio and concentration by hand.  Fortunately, the lab at school recently got a nanodrop DNA quantification machine.  To use it you just drop 2uL between the silver jaws, and hit “measure”.

The interface for the nanodrop. The prep from BOSSLAB is at the top of the queue!

It turns out our prep went OK.  The stats are:

  • A260=.887
  • A280=.675
  • 260/280 1.31
  • concentration=44ng/ul

To give you an idea of how much DNA that is (and that is quite a bit of DNA!), the plasmid used for the initial transformation was at a concentration of 5ng/ul.  So this prep is almost 9x the concentration.  The volume of DNA from Carolina was 200uL; the volume of the prep we did was (if I recall correctly) 100uL (or more).  So we have now purified 4.5x  the amount of DNA we started with!  Pretty impressive.  The question is- now what do we do with it?

4 thoughts on “GFP Project: DNA Quantification

  1. yanz says:

    There is an additional A230 option in the nano drop that I saw while using one myself.. Do you know the use of an absorbance reading at that wavelength?

    • tequals0 says:

      It is used to determine RNA purity with A260/A320:

      According to quaigen: “A230 include “salt”, carbohydrates, peptides, and phenol (or aromatic compounds in general). In our experience, the increased absorbance at 230 nm in RNA samples is almost always due to contamination with guanidine thiocyanate, present at very high concentrations in the lysis buffer or extraction reagent used in most RNA purification procedures.”

      • Job says:

        Technically, you should use both 260/280 and 260/230 ratios to measure the purity of your DNA and RNA.

        As you already mentioned, the 260/280 ratio is a measure of nucleic acid absorbance, and for high-quality DNA (free from RNA), the 260/280 ratio should be above 1.8. For high-quality RNA (free from DNA), the 260/280 ratio should be above 2.0.

        In general, as you alluded to in your comment, the 260/230 ratio is a measure of “other” contamination (including salt and other compounds, but most importantly proteins). For high-quality DNA and RNA (free from protein/other contaminants), the 260/230 ratio should be above 2.0.

        This is an extremely high number, of course, and most downstream applications don’t require nucleic acids of that high quality level. Most sequencing centers, however, recommend 260/230 ratios between 1.8 and 2.0, and at our research institute, we don’t proceed with anything less than 1.5. We’re using top-quality reagents and equipment, so you shouldn’t hold yourself to these standards, but I just wanted to clarify your comment.

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