Bio 235 Lab methods final Practice Exam

Bio 235 Lab methods final CTAB - dissolves phospholipid bilayer of cytoplasmic and organelle pitates polysaccharides in cell and isolates from DNA chloroform - dissolve lipids and precipitation polypeptides what do organic solvents do? - separate out from the aqueous phase basis of phase separation of the chloroform mix and aqueous lysate - facilitates purification of DNA from other biological macromolecules. chloroform is hydrophobic w/ everything it dissolves and separates from hydrophilic DNA differential density of chloroform and aqueous lysate in separating DNA from macromolecules - aqueous lysate is less dense and on top of org layer. makes it easier to separate how does high concentration of ions and ethanol facilitate the precipitation of DNA - ions shield neg charges on DNA. lessens natural repulsion to itself and allows it to be more tightly packed. ethanol dehydrates the solution and causes DNA to get even closer together. results in DNA pellet What provides the specificity of PCR? - -primers, they will only amplify the region of DNA we want What is the purpose of the following contents of the master mix: buffer, forward primer, reverse primer, dNTPs, and Taq polymerase? - -buffer: keeps pH low -foreword and reverse primer: prime each end of the DNA fragment needing to be copied -dNTPs: building blocks for the new strand -taq polymerase: the enzyme that is commonly employed in PCR, enzyme that copies DNA Why did not we allow the primers to anneal at room temperature? - -it allows for optimal base-pairing; room temperature is not optimal What is the purpose of the "final extension" step in PCR? - -gives Taq polymerase time to find and complete the elongation of all unfinished strands and to ensure that it will add a final dATP nucleotide to the newly synthesized stand ("overhang") Why do we use DNA polymerase (the Taq polymerase) from the bacterium Thermus aquaticus when we perform PCR? - -we use this because it can withstand the high temperatures of denaturation What are the essential enzymes that carry out DNA replication? - -helicase: unwinds DNA -primase: builds primer -polymerase: adds nucleotides -ligase: connects the backbone of DNA Be able to recognize pyrimidines, purines, nucleosides, nucleotides, NTPs, deoxyNTPs, dideoxyNTPs. - -purine: 2 circles, A&G -pyrimidine: 1 circle, CT&U -nucleoside: nitrogenous base (AGCTU) + pentose (sugar) -nucleotide: base + pentose (sugar) + phosphate group Know how to calculate the number of hydrogen bonds in a DNA molecule of known nucleotide composition. - -A-T: 2 hydrogen bonds -G-C: 3 hydrogen bonds