Extracting and Analysing Plasmid DNA From E.coli

Extracting and Analysing Plasmid DNA From E.coli Introduction Deoxyribonucleic acid (DNA) is a molecule present in all living things, and they carry genetic information which determines every characteristic a person can have. DNA contains 4 chemical units: Adenine, Guanine, Thymine and Cytosine. These 4 letters are organized to make genes which contain information to make proteins. As scientists have discovered, it is the genome (DNA sequence in a particular arrangement of the 4 letters) that makes every human unique. During the first stages of cell division, the human DNA is organized into 46 tightly coiled structures called chromosomes. As a cell divide, the chromosomes are copied over to the new cells, ensuring they receive a full copy of the genetic blueprint. Objective Isolate DNA of cheek cells Extract chromosomal DNA from strawberry Extract plasmid DNA from E.coli. General Method Collect cells Split cells open and release contents Destroy enzymes which break apart DNA Separate DNA from other cell components Precipitate DNA General Materials Solution I Solution II Solution III Tubes of various sizes according to use Proteinase K (10mg/ml) Strawberry Filter funnel DNA extraction buffer Chlorofoam LB Liquid Medium 5M NaCl 70% Ethanol 95% Ethanol Centrifuge Hot water bath Lysis Buffer DNA of Cheek Cells Collect cheek cells by rinsing mouth with saline solution Saline solution prevents cells from splitting open too soon Spin solution in a centrifuge to collect cells at the bottom of the tube Empty out the liquid, leaving the cell pellet at the bottom Add Lysis Buffer (Contains soap, salts and ions, buffers) Soap: Destroy fatty membranes that enclose cells Destroy nuclei membranes in the cells Salts and ions: Bring up osmotic pressure (pressure applied to solution needed to prevent the inflow of water) outside the cell, which helps break apart membranes Buffer: To maintain pH Breaks open cells DNA released into solution Add Proteinase K Digest contaminating proteins Degrades nucleases which attack nucleic acids Put the solution in hot water bath Enables Proteinase K to work efficiently Kill enzymes in the cytoplasm which can break apart DNA Add 5M NaCl Change polarity of solution to differentiate DNA from fats, carbohydrates and proteins DNA dissolves in ionic solutions, the rest do not Centrifuge solution Separates DNA (dissolved in clear liquid) from fats, carbohydrates and proteins (solid pellet) Transfer clear liquid (containing DNA) to new tube Add cold 95% ethanol to new tube Precipitate dissolved DNA from ionic solution since DNA is not soluble in alcohol The colder it is, the less soluble DNA (Can precipitate more) Coldness slows down enzymatic reactions which can break DNA apart Centrifuge new tube Resulting white pellet is DNA of cheek cells DNA of Strawberry Mash strawberry Add DNA extraction buffer (contains shampoo/soap NaCl) and mash Shampoo/soap: Dissolves cell membrane which is made up of lipid bilayer NaCl: Removes proteins that are stuck onto DNA Prevent proteins from precipitating along with DNA in ethanol Filter and add cold ethanol Precipitate DNA Twirl glass rod at interface between ethanol layer and slurp layer Resulting sticky mass is the plant DNA Plasmid DNA of E. coli Add solution I (contains glucose, Tris, EDTA) to prepared pellet Glucose: Increase osmotic pressure outside cells Tris: Maintain constant pH EDTA (Ethylenediaminetetraacetic acid): Protects DNA from enzymes which will degrade DNA Add solution II (contains alkali substances detergent) Alkali: Breaks open the cells Break down DNA into single strands Detergent: Break membrane apart Add solution III (contains acidic substances) Neutralizes pH so DNA strands can get back together as double stranded Precipitates cellular debris E. coli plasmid DNA remains in solution Add chloroform Extract DNA Centrifuge mixture Separates plasmid DNA and debris chromosomal DNA Transfer some amount of liquid into new tube Add 95% ethanol Centrifuge new mixture Purify plasmid DNA Pour away liquid and add 70% alcohol Remove remaining salts Centrifuge mixture Pour away liquid and spin the tube Resulting pellet is plasmid DNA Discussion/Extensions Why is DNA extraction important/used for: Crime and historical identification Lineage/origin identification Diagnosis of diseases Mass produce gene/protein important for treating diseases, using further DNA technology Genetic engineering Other DNA extraction methods: Anion-exchange Uses chromatography technique Nucleic acids of DNA are composed of negatively-charged phosphates Positively-charged substrate used to bind to the negatively-charged phosphates Proteins and RNA are then removed with medium-salt buffers Silica Gel Advantage: Fast, reliable, economical Uses silica-gel membrane to adsorb nucleic acids of DNA Catalysts: Chaotropic salts Buffers used in lysis helps DNA to adsorb on silica-gel membrane, and washes away metabolites and proteins Salting Remove proteins and contaminants by using high concentrations of salt Precipitates removed using centrifuge DNA recovered with alcohol Organic extraction Mix dead cells with phenol, chloroform and alcohol DNA extracted using alcohol precipitate Cesium chloride (CsCl) Mix suspended DNA with CsCl and ethidium bromide Solution centrifuged DNA extracted with isopropanol Limitations This general method of DNA isolation consists of many limitations: Inability to remove inhibitors of polymerase chain reaction Too many steps may be too time-consuming Multiple tube transfers may increase risk of contaminations by à ¢Ãƒ ¢Ã¢â‚¬Å¡Ã‚ ¬Ãƒâ€¹Ã…“outsideà ¢Ãƒ ¢Ã¢â‚¬Å¡Ã‚ ¬Ãƒ ¢Ã¢â‚¬Å¾Ã‚ ¢ DNA Conclusions This simple experiment provides an introduction to the procedures that are used in modern microbiological laboratories. Other cases can get much more complex, and will involve more sophisticated methods and equipment. The extraction of DNA is the first step of many other fascinating processes, which includes the manufacturing of medicines as well as genetic engineering which alters the genes of organisms.