Biology 201 Lab # 11 Fall 2017 Dr. Santos DNA extraction Biology 201 Lab # 11 Fall 2017 Dr. Santos

Deoxyribonucleic acid

Where found? Function? Structure? Composition?

DNA extraction DNA extraction is the process of obtaining pure DNA from a sample, either from living or non-living cells, such as those found in viruses. This technique is commonly used in the medical field, where early detection of diseases and disorders significantly increases the survival rates of afflicted individuals.

A; Isolation of DNA from plant tissue Very important! 5 major steps in the process of extracting DNA. 1- break the cell wall and dissolve the cell membrane to liberate the nucleus 2- isolate the nucleus from the cellular debris 3- break the nuclear membrane to release the DNA into solution 4- remove all contaminating proteins from the solution 5- protect the DNA from cellular nucleases present

Procedure 1- blend your onion specimen, 1/8 teaspoon of salt, and cold water. DO NOT COMPLETELY LIQUEFY The blending- will break apart the cell walls The salt will neutralize the negative charges on the DNA and thus enables the DNA strands to stick together. It also causes proteins and carbohydrates to precipitate.

2- filter the mixture through chees cloth or coffee filter into a glass beaker! 3- add 2 tablespoons of liquid detergent to the mixture, gently swirl, and let it stand for 5 minutes. After the cell walls have been disrupted during mechanical mashing of the onion, the liquid detergent disrupts the cell and nuclear membranes of each cell to release the DNA. It does this by dissolving lipids and proteins that hold the membranes together. Another way to disrupt the membranes is treat it with SDS (Sodium dodecyl sulfate), at 720C, a strong anionic detergent. The SDS also destroys nucleases that might destroy our DNA sample.

4- after 5 minutes, pour the mixture into 4 test tubes (1/3 of the way only) 5- add 1/8 teaspoon of meat tenderizer to each tube and gently swirl The meat tenderizer, contains proteases, breaks down the protein core, the histones, that DNA is supercoiled around. Other ways to get rid of proteins is using chloroform and “salting out” the proteins. 6- tilt each tube and slowly add cold ethanol. Let it stand for 5 minutes. Ethanol is used in DNA extraction to force the DNA to precipitate in a solution

Determination of DNA concentration DNA absorbs UV light between 250 and 280 nm due to the presence of the ring structures. When we use a spectrophotometer set at 260 nm, an absorbance of 1 = 50 µg/ml. We can estimate the concentration of DNA by measuring the absorbance at 260 nm and multiplying what we get by 50 µg/ml. Example; After extracting DNA, a student places a small sample in a spectrophotometer tube and reads the absorbance at 260 nm. The absorbance is 0.4. Determine the DNA concentration.