Help me number 3 and 4 This is the information for lab Before starting I want to

Question

Help me number 3 and 4

This is the information for lab

Before starting I want to understand.

3. With the knowledge that SDS is a strong denaturing detergent (it is amphipathic, meaning it has a high affinity for binding to both polar and nonpolar molecules), how might it work in concert with proteinase K to free DNA bound to histone proteins within the nucleosome/chromatosome core? Also, what does this say about PK? 4. As noted, addition of ethanol to aqueous DNA pulls or "steals" water molecules from around the dissolved DNA, forcing it out of solution. NaOAc is another component of the Precipitation mix in addition to ethanol. How might DNA precipitation on a molecular level? this salt further this process of

Explanation / Answer

3. For Bacterial and Human genome isolation SDS-protein kinase K is a suitable method. Sodium dodecyl sulfate (SDS) is a negatively charged or anionic detergent used in protein denaturation and lyse the cell membranes. Proteinase K, is a serine protease, works better in presence of a detergent (like SDS) or a chaotropic salt (guanidinium isothiocyanate) and at elevated temperatures. This enzyme cleaves the peptide bond in proteins next to the carboxyl group of hydrophobic amino acid residues (both aliphatic and aromatic). High temperatures, detergents, and chaotropic salts help to denature proteins, exposing many more hydrophobic amino acid residues that would normally be hidden in the hydrophobic core of the protein. This step is necessary for lysis of the cells and nuclei and liberates the DNA tightly bound in chromatin.

4. Alcohol precipitation is the most reliable method of nucleic acid precipitation. Nucleic acids are hydrophilic due to the negatively charged phosphate (PO3-) groups along the sugar phosphate backbone. Water has a partial negative and positive charges on oxygen and hydrogen respectively. They may interact with DNA phosphate (PO3-) group and solubility may increase. To avoid this method and easily pool out the DNA we have to depend on NaOAc (sodium acetate). The salt which is commonly used to extract DNA is sodium acetate. When it hydrolyse, it breaks up to Na+ and [CH3COO]- ions. Sodium ions nullify the negative charge on the PO3- groups on the nucleic acids and make it less hydrophilic in nature and thus less soluble in water.

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