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”.
It turns out our prep went OK. The stats are:
- 260/280 1.31
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?