Determination of DNA concentration and purity
Ethidium bromide (EB) is an embedding dye with a phenanthridine ring that can be inserted between the stacked bases of a DNA or RNA strand. The proximity of the embedding group of ethidium bromide to the bases makes the two bind tightly.
The DNA absorbs UV radiation at 254 nm and transfers energy to the EB, which itself absorbs light at 302 nm and 366 nm. The absorbed energy is released at 590 nm and appears as orange-red fluorescence. The fluorescence yield of bound EB is much higher than that of free EB.
The amount of EB bound is related to the size and configuration of the DNA, and the fluorescence intensity is proportional to the amount of ethidium bromide embedded. For homogeneous DNA samples of a single conformation, the amount of ethidium bromide embedded is proportional to the DNA content of the sample solution.
Operation method
ethidium bromide method
Principle
Ethidium bromide (EB) is an embedding dye with a phenanthridine ring that is inserted between the stacked bases of a DNA or RNA strand. The proximity of the embedding group of ethidium bromide to the bases causes the two to bind tightly together.The DNA absorbs UV radiation at 254 nm and transfers the energy to EB, which itself absorbs light at 302 nm and 366 nm. The absorbed energy is released at 590 nm and appears as orange-red fluorescence. The fluorescence yield of bound EB is much higher than that of free EB, and the amount of EB bound is related to the size and conformation of the DNA, while the fluorescence intensity is proportional to the amount of embedded ethidium bromide. For homogeneous DNA samples of a single conformation, the amount of ethidium bromide embedded is proportional to the DNA content of the sample solution.
Materials and Instruments
Standard DNA samples (linearized DNA of known concentration, a series of standard samples diluted in TAE to gradient concentrations) Move 1. Spot two rows of ethidium bromide 2 μl droplets on the black plastic plate, six spots in each row. 2, take 2 μl of standard sample and mix with the first row of ethidium bromide, so that the DNA concentration of each point is 20, 15, 10, 5, 2, 1 (unit: ng/μl). 3, take the sample to be tested after gradient dilution, each add 2 μl in the second row of ethidium bromide and mix well. 4. Put the above plastic plate on the sample stage of the UV projector and close the door of the sample room. Excite the fluorescence with short-wave UV light and observe the fluorescence intensity of each point or take a picture. 5. Compare the brightness of the upper and lower rows to determine the concentration of the sample to be tested. Caveat 1. The standard sample contains a single type of DNA and is similar in size to the sample to be tested. 2. The same volume is used for the sample to be tested and the standard sample. 3， EB is moderately toxic and highly carcinogenic, remember to wear gloves during operation and do not contaminate clothing, skin, eyes, mouth and nose. 4、EB-stained utensils should be concentrated in the recycling container after use, and should be decontaminated before disposal. 5、This method can only estimate the DNA concentration of the sample, can not get the specific concentration, it is more recommended to use Nanodrop 2000 to determine the DNA concentration. Common Problems 1. How to choose a suitable standard sample size? A. Through the pre-test, choose a large gradient standard sample to estimate the approximate size of the sample to be tested, and then reduce the gradient of the standard sample to carry out the formal experiment. For more product details, please visit Aladdin Scientific website.
Enzymatic samples of plasmid DNA (to be tested)
Ethidium bromide EB 5 μg/ml (prepared by diluting 10 mg/ml master mix with TAE at pH 8.0)
UV transilluminator