CHEM 2720 Experiment 6 PDF

CHEM 2720 EXP 6 week 1Important info Lysate- fluid containing lysed cells Cell lysis breaks down cells and releases cellular components such as organelles, cytosol, dissolved salts, etc in an aqueous mixture known as lysate. Ways to lyse a cell? 1. Grind in mortar and pestle 2. freeze/thaw...

CHEM 2720 EXP 6 week 1Important info Lysate- fluid containing lysed cells Cell lysis breaks down cells and releases cellular components such as organelles, cytosol, dissolved salts, etc in an aqueous mixture known as lysate. Ways to lyse a cell? 1. Grind in mortar and pestle 2. freeze/thaw in liquid nitrogen 3. Sonification by high-frequency waves 4. Membrane solubilization by detergents How do you lyse bacterial cells such as E.coli? 1. Enzymatic degradation of the cell wall by an enzyme lysozyme can be used. Lysis can be performed at room temperature or at 4 degrees Celsius. Why can purification of nucleic acids be performed at room temperature? Because the harsh conditions of cell lysis damage cellular structures and denature nucleic acid, degrading enzymes will leave nucleic acids intact or in a state from which their native structure can easily be recovered. To sum it up, it minimizes the deterioration of the protein. What are proteases? Enzymes that cut proteins into smaller pieces. Most commonly used methods of chromatographic separation: 1. Size exclusion chromatography 2. Ion-exchange chromatography 3. High-performance liquid chromatography 4. Capillary electrophoresis How to use kits to extract and purify nucleic acids: after cells of tissue have been lysed, the kits work by selectively degrading and precipitating non-nucleic acid biological molecules which are removed by centrifugation or by washing them off a column. What is electrophoresis? a way to separate charged molecules in an electric field. Used to separate different nucleic acid molecules depending on the size. Commonly used electrophoresis is agarose gel electrophoresis which can be used to partially purify nucleic acids, how? After you run the nucleic acids from the gel, you can physically cut the piece of gel that contains the nucleic acid you want and then purify it further using spin column technology. What is the drawback of purifying using this method? You cannot separate them from each other as they will both migrate to the same place on the gel. How do we fix that? Using recombinant nucleic acid techniques ensures you only have a single type of nucleic acid molecule of that size. How to quantify purity for enzymes? Determine specific activity. What is specific activity? The ratio of the amount of enzyme activity that is present in a sample to the amount of protein. How do we quantify protein activity? We add a fixed volume of the purified product to an excess of the enzyme-substrate and then the amount of product that is formed overtime i measured with a spectrophotometer. Most common way to evaluate the purity of a protein preparation? Sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE). What does it do? It separates different proteins based upon the mass of their polypeptide chains on a porous gel under the influence of an electric field. If different sized proteins are present in the sample ( it is not pure) multiple bands or a smear will appear on the gel when visualized. A pure sample should only produce a single band. What are plasmids? A class of small, non-genomic double-stranded nucleic acids, usually DNA, that can be found in several domains on microorganisms. They belong to the genetic element family known as replicons. What are replicons? Units or DNA or RNA capable of replicating independently Plasmids that carry genetic information favourable to the host cell, such as antibiotic resistance. This will promote their spread to daughter cells and the plasmic will generally be retained by the host so long as they confer a survival advantage to the host. Plasmid contains genetic information that promotes their direct transfer between different individual microorganisms using mechanisms such as conjugation, transduction, or transformation. Purification of plasmic from E.coli is achieved through a series of procedures collectively known as? Mini or maxi prep which include: resuspension, lysis, neutralization, binding, washing, and elution. Resuspension: E.coli is harvested by centrifugation, growth media is removed and the pellet is resuspended in a mixture of TRIS, EDTA, glucose, and RNAase. What does the TRIS do? Maintains pH. What does EDTA do? Sequester cations that are necessary for the activity of DNA nucleases, ensure no catalytic degradation of DNA occurs. What does glucose do? Maintains the osmotic strength of the solution to prevent premature cell degradation. What does RNAase do? Degrades RNA. Lysis: cell rupture is achieved by the addition of the lysis buffer that contains NaOH and SDS. Causes rupture of the cell, and the high pH and the detergent effect of the SDS degrade proteins causing them to form aggregates and come out of the solution; resulting in the denaturation of of DNA into single-stranded forms. Neutralization: What does the addition of a neutralization solution do? Promotes re-annealing of the DNA that was denatured in the lysis step. Since plasmic DNA is small in comparison to genomic DNA it is able to properly base pair thereby reforming double-stranded circular plasmid. Genomic DNA cannot properly reanneal so it retains a disordered structure and remains insoluble in the condition of the neutralization solution. Plasmid and Genomic DNA are separated through centrifugation, plasmid remains in the supernatant and the non-plasmic DNA in the pellet. Binding: Plasmic DNA from supernatant is applied to the silica-based spin column that comes with the kit and the high salt conditions promote the formation of salt bridges between the sugar-phosphate backbone of the DNA, and the silica membrane of the spin commun thereby capturing the DNA on the membrane. Washing: DNA bound to the silica membrane is washed severally with an ethanol-rich solution. Why? To rinse away contaminants and excess salts leaving isolated DNA bound to the membrane. Elution: DNA is removed from the column into a microfuge tube by adding elution buffer, which has low salt content, and is properly buffered to maintain DNA integrity. When adding Lysis solution to Ecoli why is it important to mix gently? If mixed vigorously, there is a risk of shearing the chromosomal DNA which will then make it difficult to separate plasmid DNA. Why is it important to leave behind a little supernatant? As a safely precaution to ensure you do not transfer any pellets. How was spinach DNA lysed in the experiment? It was ground into a fine powder using mortar and pestle and liquid nitrogen. Exp 6 week 2