When calculating DNA concentration, why must we multiply by a dilution factor?

Hello! Ok, the OD260 stands for optical density, and is a measure of the light absorbency of DNA or RNA. So what you did to get this number was extract DNA (or RNA, but I'll use DNA as an example) from a cell, then dilute it in order to get enough volume to read the OD260 without using up your whole sample.

In general, we use 10 ul in 40 ul of buffer, for a dilution of 1:5. We then get the OD260 reading, which is a reading of how much light is absorbed by our solution, which gives us an idea as to how much DNA is present in the sample. In order to change this OD reading from an absorbency number to something that tells us how much DNA is there, we use the conversion factor:

A260 OD 1 = 50 ug of dsDNA/ml

This means for every 1 OD260 reading, we have 50 micrograms of DNA per milliliter. So you have to multiply your OD260 number by 50 in the case of double stranded DNA, by 40 in the case of RNA, and by 33 in the case of single stranded DNA just to transform the number your equipment gave you into how much DNA you have in your sample.

Then you have to multiply by the dilution factor to figure out how much DNA is present in your original, undiluted sample.

To answer your question more concisely, multiplying by 50 changes the number from the spectrophotometer into how much DNA is present in the diluted sample, while multiplying by the dilution factor tells you how much DNA is in the undiluted sample.

I hope this helps!

A spectrophotometer measures the amount of light that passes through a sample. Nucleic acids like DNA and RNA contain nitrogenous bases that absorbs ultraviolet light. The more DNA or RNA in a sample, the more UV light is absorbed. You can calculate the concentration of DNA and RNA in a sample by comparing how much UV light is absorbed compared to a blank sample. The wavelengths used for this technique is 230nm, 260nm and 280nm.

To calculate the concentration of DNA or RNA, you multiply the absorbance at 260nm by the appropriate factor. Because the nitrogenous bases in double stranded DNA are buried in the middle of the double helix, it takes more dsDNA to make one unit of absorbance. In single stranded DNA, the bases are unobstructed so they can absorb light easier so it takes less ssDNA to cause the same amount of absorbance (50 ng/ul/abs vs. 33 ng/ul/abs). ssRNA is in between dsDNA and ssRNA and it absorbs UV260 at 40 ng/ul/abs.  

The ratio of absorbance of 260/280 indicates how pure your sample is. This ratio should be between 1.8-2.0 for DNA and 2.0 or greater for pure RNA. The 230/260 ratio should be 2.0 or greater. A low 230/260 ratio could mean there is phenol contamination in the sample.

To make this measurement, you have to use your RNA or DNA that you purified. Unless you have a Nanodrop (which only uses 1 ul of sample per measurement) you need to fill a UV cuvette with a large volume. It would be a waste of your precious sample to use up all of it to measure its Abs260. Instead, you add just a little bit of it to a large volume of diluent. By diluting your sample, you only use a small amount of the nucleic acid you purified. But, when you measure the concentration, you are not measuring the concentration of your sample, instead, you are measuring a diluted sample. To figure out the original concentration, you multiply your reading by the amount of your dilution.