DNA quantity and quality

…there seems to be confusion as to how to best *estimate* the concentration and quality of DNA. I summarise which method you should use when! It is really difficult to get an *absolute* measurement but you *must* use the most appropriate method to get the best estimate. 

Agarose gel (stained SYBRSafe or rarely ethidium Bromide)- quantity and physical quality of DNA (using known lambda DNA standards held in the lab stock). RNA will fluoresce. Use on DNA from all tissue types.

Nanodrop (no dye- it is a spectrophotometer)- quantity and purity (salts etc. and proteins) for both DNA and RNA solutions. Both absorb maximally at 260nm so you cannot tell how much of each in a mixed sample. Use when you know there is no RNA in the DNA solution or vice versa (as a guide bird blood is usually OK- tissue samples are not- if you don’t know run on an agarose gel 1st).

Fluorometer-(Hoechst33258)-  asesses the quantity of DNA only (binds to AT- calf thymus DNA standard which we use has an AT of 58% which should reflect your organism- if it doesn’t you will need to do an adjustment calculation- it is good for most plant and animal DNA). Take care when quantifying DNA with difft. conformation from the standard (SS DNA, plasmids).

 hope that helps!

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