The relatively simple composition of extraction buffer A performs a rather compl
ID: 82142 • Letter: T
Question
The relatively simple composition of extraction buffer A performs a rather complex function in this experiment. Provide answers to the following in order to describe how the extraction buffer isolation of acids. a. List the main components of extraction buffer A and briefly describe the physical properties of these components. b. Although the cell membrane and nuclear envelope are destroyed during the pulverizing step, how are these membrane molecules separated from the nucleic acids. What happens to these components after being separated from DNA? There are many proteins embedded in nucleic acids. a. What are these proteins called? b. How are these proteins separated from the DNA? c. What happens to these proteins after being separated from DNA?Explanation / Answer
1)
a) Since extraction buffer solution A is not given in the question, I will describe about the composition of extraction buffer that we normally use for extraction of nucleic acids.
1. Extraction(CTAB) Buffer
2. Isopropanol
3. Saturated phenol pH 8.0
4. Chloroform : isoamylalcohol ( 24:1) mixture
5. Tris:EDTA
6. RNase
7. 70% ethanol
Cetyl trimethyl ammonium bromide (CTAB) or SDS disrupts the membranes, B mercaptoethanol acts as a reducing agent, which helps in denaturing proteins by breaking the disulfide bonds between the cysteine residues and for removing the tanins and polyphenols present in the crude extract, EDTA acts as a chelating agent which chelates the magnesium ions required for DNase activity, Tris serves as buffer at pH 8 and sodium chloride aids in precipitation by neutralizing the negative charges on the DNA.
b) Cellular and histone proteins bound to the DNA can be removed by adding a protease or by precipitating the proteins with sodium or ammonium acetate, or extracting them with a phenol-chloroform mixture prior to the DNA-precipitation.