DNA Spectrophotometry

Question 1

Classic Spectrophotometers

Answer 1

Analog
Digital

Question 2

DNA has its absorbance maximum at ______

Answer 2

260 nm

Question 3

Spectrophotometer Cuvettes

Answer 3

Optical glass
ES Quartz
IR Quartz
PS or PMMA

Question 4

Spectrophotometry (UV-Vis)

Light source -> Sample -> Detector

Question 5

Pure Nucleic Acid solution

A260/A280 = ~1.8 (Pure DNA)
A260/A280 = ~2.0 (Pure RNA)

Values < 1.8, indicates contamination probably caused by organic compounds or chaotropic agents, which absorb at 230 nm.

Question 6

Protein absorbance maximum is at _______

Answer 6

280 nm

Question 7

Possible contaminants in spectrophotometer

Answer 7

Sugar, salts, and organic solvents

Question 8

DNA Quantitation Using a Spectrophotometer
Video: UV-Vis Spectrophotometry

1. Turn on spectrophotometer 10 minutes before starting so it has time to warm up
2. Prepare 2 cuvettes that are transparent to UV light. When handling cuvettes, always place your fingers on the opaque side and not on the transparent surfaces of the cuvette.
3. Label 1 on the cuvettes C for control and the other cuvette will be your test.
4. Set your micropipette to 245 microliters.
5. Pipette 245 microliters of dH2O into both cuvettes.
6. Set your micropipette to 5 microliters.
7. Pipette 5 microliters of purified DNA sample into the test cuvette.
8. Pipette up and down to mix the sample.
9. Set the spectrophotometer to its DNA quantitation setting.
10. Your screen should show that you have selected for DNA.
11. Press the enter button twice.
12. The display screen should now show that you are ready to read the absorbance.
13. Press the right arrow button. Insert the control cuvette, making sure the correct side is facing forward.
14. Press the read blank button. After completing this process, the screen should read 0 absorbance.
15. Remove the control cuvette and insert the test cuvette with the DNA sample.
16. Press the read sample button and then hit the right arrow. When finished, the display will show that the absorbance is 0.011 and the concentration is 0.5415 micrograms per milliliter.

The original dilution was 50 to 1 so you will have to multiply by 50 to get the final concentration.

17. To get the purity, press the 260 to 280 ratio.

The result here is 5.4921. Anything greater than 1.8 is pure DNA. Therefore in this case, we have determined that we have purified plasmid

Question 9

How to Quantify DNA with a Spectrophotometer
Video: Nanodrop

1. Run a blank using the same solution as you used for the dilution of the DNA, in this case, it’s water. Put 1.5 microliters of water and click blank.
2. After the blank is made, load the sample but before that, wipe the measuring area with a clean tissue and load 1.5 microliters of the sample. Make sure that there’s no bubbles.

We can see that there is a prominent peak at 260 nm which corresponds to the DNA and the concentration that is measure at this peak is around 200 nanograms per microliters.

3. Also look at peaks around 230 and 280 nm because these corresponds to wavelengths at which possible impurities will absorb. In this case, if there are no peaks then the DNA is pure and the concentration corresponds to the concentration of the DNA.

One common mistake is to measure DNA concentration from the unpurified PCR sample (results are = there is a peak at 230 nm and shifted towards 280 nm - corresponds to impurities. Also, even if the concentration is measured, you cannot take this concentration as the actual concentration of your amplified DNA in the mix because not only amplified DNA strands will absorb at this wavelength but also unused dntps which means that this concentration does not to the concentration of your PCR product.

Question 10

Nanodrop is also called

Answer 10

Tabletop spectrophotometer

Question 11

NanoDrop Microvolume Quantitation of Nucleic Acids
Video: Nanodrop

1. 2.

Question 12

Is innovative technology that uses the inherent surface tension of liquids to hold and measure microvolume samples between 2 optical pedestals without the use of cuvettes or capillaries.

Answer 12

Nanodrop Sample Retention System

Question 13

As little as 1 microliter of sample is pipetted directly on top of the lower optical surface. Upon closing a spectrometer arm, a liquid column is created between the upper and lower optical pedestals by surface tension, forming a vertical optical path. The vertical path length can automatically change in real time during the measurement, shortening the path length enables the measurement of higher concentrations, effectively removing the need to perform dilutions for most nucleic acid samples. Furthermore, the sample retention system expedites the experimental process with a fast cleanup which consists of simply wiping optical surfaces with a laboratory wipe.