Extracting and Analysing Plasmid DNA From E.coli

Extracting and Analysing Plasmid desoxyribonucleic acid From E.coliIntroductionDeoxyribonucleic acid (desoxyribonucleic acid) is a molecule present in all living things, and they carry genetic tuition which determines every characteristic a person can have. deoxyribonucleic acid 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 benevolent 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 assume of the genetic blueprint.ObjectiveIsolate DNA of nervus cellsExtract chromosomal DNA from strawberryExtract plasmid DNA from E.coli. world(a) Method forgather cellsSplit cells open and rel ease contentsDestroy enzymes which break apart DNASeparate DNA from other cell componentsPrecipitate DNAGeneral MaterialsSolution ISolution IISolution IIITubes of various sizes according to useProteinase K (10mg/ml)StrawberryFilter funnelDNA extraction bufferChlorofoamLB Liquid Medium5M NaCl70% Ethanol95% Ethanol centrifugateHot water bathLysis BufferDNA of Cheek CellsCollect cheek cells by rinsing mouth with saline solutionSaline solution prevents cells from splitting open too soon rotate solution in a centrifuge to collect cells at the freighter of the tubeEmpty out the liquid, leaving the cell pellet at the bottomAdd Lysis Buffer (Contains soap, salts and ions, buffers)Soap Destroy fatty membranes that enclose cellsDestroy nuclei membranes in the cellsSalts and ions Bring up osmotic pressure (pressure applied to solution compulsory to prevent the inflow of water) outside the cell, which helps break apart membranesBuffer To maintain pHBreaks open cellsDNA released into solution Add Proteinase K tin contaminating proteinsDegrades nucleases which attack nucleic acidsPut the solution in hot water bathEnables Proteinase K to work efficientlyKill enzymes in the cytol which can break apart DNAAdd 5M NaClChange polarity of solution to differentiate DNA from fats, carbohydrates and proteinsDNA dissolves in ionic solutions, the rest do notCentrifuge solutionSeparates DNA (dissolved in clear liquid) from fats, carbohydrates and proteins (solid pellet)Transfer clear liquid (containing DNA) to new tubeAdd cold 95% ethyl alcohol to new tubePrecipitate dissolved DNA from ionic solution since DNA is not soluble in alcoholThe colder it is, the less soluble DNA (Can precipitate more)Coldness slows down enzymatic reactions which can break DNA apartCentrifuge new tubeResulting white pellet is DNA of cheek cellsDNA of StrawberryMash strawberryAdd DNA extraction buffer (contains shampoo/soap NaCl) and mashShampoo/soap Dissolves cell membrane which is made up of lipid bilaye rNaCl ingests proteins that are stuck onto DNAPrevent proteins from precipitating along with DNA in fermentation alcoholFilter and add cold ethanolPrecipitate DNATwirl glass rod at interface between ethanol layer and slurp layerResulting sticky mass is the go under DNAPlasmid DNA of E. coliAdd solution I (contains glucose, Tris, EDTA) to prepared pelletGlucose Increase osmotic pressure outside cellsTris Maintain constant pHEDTA (Ethylenediaminetetraacetic acid) Protects DNA from enzymes which will degrade DNAAdd solution II (contains alkali substances detergent)Alkali Breaks open the cellsBreak down DNA into single strands purifying Break membrane apartAdd solution III (contains acidic substances)Neutralizes pH so DNA strands can get back together as double strandedPrecipitates cellular debrisE. coli plasmid DNA remains in solutionAdd anesthetiseExtract DNACentrifuge inter compartmentalizationSeparates plasmid DNA and debris chromosomal DNATransfer some amount of liquid into new tubeAdd 95% ethanolCentrifuge new mixturePurify plasmid DNAPour away liquid and add 70% alcoholRemove remaining saltsCentrifuge mixturePour away liquid and spin the tubeResulting pellet is plasmid DNADiscussion/ExtensionsWhy is DNA extraction important/used for evil and historical identificationLineage/origin identificationDiagnosis of diseasesMass produce gene/protein important for treating diseases, using further DNA technology genetical engineeringformer(a) DNA extraction systemsAnion-exchangeUses chromatography techniqueNucleic acids of DNA are composed of negatively-charged phosphatesPositively-charged substrate used to bind to the negatively-charged phosphatesProteins and RNA are then aloof with medium-salt buffersSilica GelAdvantage Fast, reliable, economicalUses silica-gel membrane to adsorb nucleic acids of DNACatalysts Chaotropic saltsBuffers used in lysis helps DNA to adsorb on silica-gel membrane, and washes away metabolites and proteinsSaltingRemove proteins and c ontaminants by using high concentrations of saltPrecipitates removed using centrifugeDNA recovered with alcoholOrganic extractionMix dead cells with phenol, chloroform and alcoholDNA extracted using alcohol precipitateCesium chloride (CsCl)Mix suspended DNA with CsCl and ethidium bromideSolution centrifugedDNA extracted with isopropanolLimitationsThis general method of DNA isolation consists of many limitationsInability to remove inhibitors of polymerase chain reactionToo many steps may be too time-consuming eight-fold tube transfers may increase risk of contaminations by outside DNAConclusionsThis 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 enthralling processes, which includes the manufacturing of medicines as well as genetic engineering which alters the genes of organisms.