I am trying to verify DNA concentrations from a spectrophotometer, and then calculate yield. I took 2uL of DNA+Buffer solution (total sample volume = 100 uL) and added that to 98uL of water to dilute the sample for reading in the spectrophotometer, which provides abs. readings at 260nm, 280nm, and 320nm. What dilution factor should be used to calculate concentration ((260-320)*dilution factor*50ug/ml) ? DF = 1?
Yield in ug = sample volume in mL * concentration, yes?
Dear Lana Ruvolo,
1. You have a stock sample (100 uL)
2. You took 2 ul out of the stock sample and mixed it with 98 ul water. So, you diluted them to: [ 2uL/(2 uL + 98 uL) = 1/50x ]. In other words, the dilution factor (DF) is 50
3. You then measured the concentration of the diluted sample. Lets say that the measured DNA concentration is 10 ng/uL.
4. Then, your stock sample's concentration will be 10 ng/uL x 50 (DF) = 500 ng/uL
5. Now, you know that your stock sample's concentration is 500 ng/uL and its volume is 100 uL; therefore, the total yield (of the DNA in the stock sample) is: 500 ng/uL x 100 uL = 50,000 (ng) = 50 (ug)
6. If your department has a NanoDropTM machine, it will make your life much easier. The NanoDropTM machine can directly read you the DNA concentration for you (with only 1 drop (or 1 uL) DNA sample).
7. NanoDropTM machine will also tell you the ratio of absorbance at 260 nm and 280 nm which are used to assess the 'purity' of your DNA sample. A ratio of ~1.8 is generally accepted as “pure” for DNA sample. DNA purity is important for your downstream experiments.
dilution factor is 50. because you have diluted 2uL sample to 100uL. For example, m=2uL*C1 . m=100uL*C2 . m is same in both formula.
so 2uL*C1=100uL*C2 where C1/C2=50. hope it is clear.
Dear Lana Ruvolo,
1. You have a stock sample (100 uL)
2. You took 2 ul out of the stock sample and mixed it with 98 ul water. So, you diluted them to: [ 2uL/(2 uL + 98 uL) = 1/50x ]. In other words, the dilution factor (DF) is 50
3. You then measured the concentration of the diluted sample. Lets say that the measured DNA concentration is 10 ng/uL.
4. Then, your stock sample's concentration will be 10 ng/uL x 50 (DF) = 500 ng/uL
5. Now, you know that your stock sample's concentration is 500 ng/uL and its volume is 100 uL; therefore, the total yield (of the DNA in the stock sample) is: 500 ng/uL x 100 uL = 50,000 (ng) = 50 (ug)
6. If your department has a NanoDropTM machine, it will make your life much easier. The NanoDropTM machine can directly read you the DNA concentration for you (with only 1 drop (or 1 uL) DNA sample).
7. NanoDropTM machine will also tell you the ratio of absorbance at 260 nm and 280 nm which are used to assess the 'purity' of your DNA sample. A ratio of ~1.8 is generally accepted as “pure” for DNA sample. DNA purity is important for your downstream experiments.
If you don't have a Nanodrop, and you want to use a conventional spectrophotometer to check the purity of your DNA sample. Attached is an article for you to follow (from Promega).
It also depends on what kind of DNA you are working with and measuring--single stranded DNA (ssDNA) or double-stranded DNA (dsDNA). The number used to plug into the fomula will be different:
1 O.D. at 260 nm for double-stranded DNA = 50 ng/ul of dsDNA
1 O.D. at 260 nm for single-stranded DNA = 20-33 ng/ul of ssDNA
1 O.D. at 260 nm for RNA molecules = 40 ng/ul of RNA
The required concentration of DNA, depends on the type of DNA you are analyzing as suggested by Yuan-Yeu Yau above. Calculating DNA yield has to take into account the possible contaminants. Some of the major contaminants are proteins/salts, RNA as well as Turbidity (which most of the people neglect). Ratio of absorbance at 260 nm to that of absorbance at 280 nm will give you results. Good quality DNA will have A260/A280 ratio between 1.7 to 2.0.
DNA purity (A260/A280) = (A260 reading – A320 reading) ÷ (A280 reading – A320 reading)
Concentration (µg/ml) = (A260 reading – A320 reading) × dilution factor × 50µg/ml
In your case the dilution factor is 50 (C1V1 = C2V2)
DNA yield (µg) = DNA concentration × total sample volume (ml)
The following points should be kept in mind as summarized below -
1. Reading at 320 nm indicates the presence or absence of turbidity, another possible indication of contamination. Hence, the calculations are conducted after subtracting the A320 value.
2. Strong absorbance reading at 230 nm indicates presence of organic compounds or chaotropic salts. Hence, A260/A230 if greater than 1.5 or 1.6, then the sample is good one. Lower the ratio, more the contamination.
3. Taking a spectrum of reading from 230 nm to 320 nm will be more informative, however a simple 260/280 ratio will also do good.
Alternatively, fluorescence methods that make use of fluorometers and fluorescent DNA-binding dyes can also be used which is more sensitive as compared to the traditional absorbance method. Microplate fluorometers can provide you with enhanced option of studying DNA in multi well plates.
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