Analysing isolation of DNA plasmid and Agragose of gel electophoresis

Analysing isolation of deoxyribonucleic hot plasmid desoxyribonucleic acid and Agragose of gelatin electophoresis foot(a) The aim of this experiment was to successfully isolate a deoxyribonucleic acid plasmid from E.Coli cells (Escherichia coli). We indeed use unremarkably performed a method commonly used in biochemistry and molecular biology called agarose gel dielectrolysis. This is used to freestanding deoxyribonucleic acid and ribonucleic acid fragments according to length ar used to estimate the surface and charge of the deoxyribonucleic acid and RNA fragments or to weaken protein by size.In this procedure as stated above, we used e.coli as these are plasmid containing cells. These cells were set in a soften and flux with a solution of 1% (w/v) SDS (sodium dodecyl sulphate) which was mixed with sodium hydroxide. The alkaline solution (12.6PH) causes the molecular weight increases this causes it to become worry chromosomal DNA. utilise alkaline lyses is based on differential denaturation of chromosomal and plasmid DNA in order to separate the two. The double stranded plasmid and chromosomal DNA is converted to single stranded DNA imputable to the lyses of the cells which solubilises protein and denatures the DNA.Subsequent neutralization is potassium acetate al humbleds only covalently closed DNA plasmid DNA to reanneal and run solubilized. Chromosomal and plasmid DNA precipitate in a complex formed with potassium and SDS which is come inback(a) by centrifugation. Protein dodecyl sulphate complexes are precipitated die to it being insoluble in water. When centrifugation neutralizes the lysine it yields to a minuscule supernatant fraction that contains plasmid DNA a network of chromosomal DNA and proteinPlasmid DNA is punishing by from the supernatant by ethanol precipitation. Plasmid DNA isolated by alkaline lyses is fitted for most analyses and cloning procedures with show up further katharsis however if the isolated plasmid DNA i s sequenced and additional purification step such as phenol extraction is used.(b) The aim of Agarose gel electrophoresis is to analyse the plasmid DNA that was extracted from the procedure before. The technique of electrophoresis is based on the fact that DNA is negatively charged at neutral pH due to its phosphate backb oneness. And like any other biological macromolecules seat move within an electrical field. The rate of the DNA slows down when its moves towards opposite poles because of the agarose. The agarose gel is a buffer solution this is used to maintain the required pH and salt concentration. The agarose forms hole or wells in the buffer solution and the DNA inserted in through the holes to move toward the positive pole. As mentioned before the agarose gel slows down the rate of DNA so the littler DNA moves faster than the large molecules of DNA as the small(a)er ones fit through the whole easier. This causes the DNA to be separated by size and clear be seen visually . To make the electrophoresis to function and separate DNA molecules it must contain an electrophoresis chamber.and billet supply, combs which are placed in the chamber this is how wells are formed when agarose is placed in the gel, Trays that contains a special gel that comes in many sizes and and have UV-properties combs which is how wells are formed when agarose is placed in the gel, Electrophoresis buffer, Loading buffer, which has a thick consistancy (e.g. glycerol) so the DNA screwing be easily placed in the wells and one or two tracking dyes, these travel in the gel and help visualize how the process is being carried out and to moniter how far electrophoresis belowgone. Ethidium bromide, is a dye used to filthiness the nucleic acids.. Tran illuminator(an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.Method for plasmid isolation1.5 ml of culture that contains E.coli cells containing the plasmid pUC118 was inserted into an Eppendorf thermionic valve.This was at that placefore cartridge extractord at 13000 rpm for two transactionsThe liquid contained in the Eppendorf tube was discarded carefully by using a pipette and then inverting the tube on a test tube to remove remaining drops of the liquid without removing the bacterial pellet200 micro-liters of solution A was added to the bacterial pellet. This ensured that the suspension is homogenized (mixtures are well separated400 micro-liters of solution B was then added and mixed well these solutions contain the SDS and sodium hydroxide. This neutralizes the solutions300 micro-liters of solution C which contains the potassium acetate which was alike mixed and then was incubated on ice for 10 minutesThis mixture was the centrifuged at 13000rpm for 5 minutes750 micro-liters of this supernatant was transferred to a sweet Eppendorf tube whilst ensuring none of the precipitate was interfered with10 micro-liters if RNAse solution was added to a duplicate tube and l abeled as R+450 micro-liters of isopropanol was added to each test tube and mixed wellThis was then centrifuged at 13000rpm for 5 minutesThe supernatants were then carefully take and the DNA was contain400 micro-liters of ethanol was added and allowed to stand for a minute it allow the salts to dissolve the liquid was carefully removed so as not to remove the DNA precipitate.The sample was then allowed to dry at mode temperatureEach pellet was then dissolved in 10 micro-liters of TE bufferQ1 The viscosity afterwards 400 micro-liters of solution B was added and mixed a low viscosity was ascertained as it had a very watery texture.Q2 there was no viscosity after the transfer of 750 micro-liters of supernatant to a new eppendorf(a) Agarose gel electrophoresisThe sample obtained from the experimental procedure above were then examined using the method of agarose gel electrophoresisThe RNAse treated and untreated plasmids were examined.10 micro-liters of loading buffer was added to 1 0 micro-liters of DNA for each sampleThe samples containing DNA mixed with loading buffer were then pipetted into the sample wells, and a current was applied. This was carried out for 30 minutesIt was clear that the current was flowing as bubbles were observed to be coming clear up the electrodes.The negatively charged DNA transmigrated towards the positive electrode at the distal end, (which is usually colored red)It was analyzed that the smaller DNA molecules travelled quickly through the gel which showed that the procedure was carried out successfully as the DNA was separated according to sizeResults/ Discussion(a) Isolation of DNA plasmidThe DNA plasmid was successfully extracted from the E.coli cells and then the DNA was the successfully separated according to size by using the agarose gel electrophoresis method.Solution A contains 25 mM of Tris-HCL (pH 8.0)50 EDTA. Tris is a buffering agent this maintains a constant pH. The EDTA is used to harbor the DNA from DNAses which a re degradative enzymes the EDTA also binds divalent cations that are necessary for DNAse activity. The solution B contains SDS which is a detersive and NaOH. This neutralizes the solution, the alkaline mixture also causes the cells to rupture and the SDS the lipid membrane is broken a billet and the cellular proteins are solubilized, NaOH converts the DNA into a single strands which is caused by denaturation. The solution C contains potassium acetate (pH 4.3) the acetic acid neutralizes the pH, allowing the DNA strands to renature. The potassium acetate is added its causes the SDS to precipitate, along with the cellular debris. TheE. coli chromosomal DNA is also precipitated. The plasmid DNA remains in the solution. The viscosity of this is very high as it has a very gel like texture.When the supernatant is placed in a new eppendorf tube after 5 minutes of centrifuge this causes the plasmid DNA to separate from the cellular debris and chromosomal DNA in the pellet.The isopropanol is then added this pulls the plasmid out and causes it to precipitate nucleic acids. after centrifuge a small white pellet was observed at the bottom of the tube after the supernatant was carefully removed this further purifies the plasmid DNA from contaminants.400microliters of ethanol was added this washed the residual salt and SDS from the DNA.All these changes that were observed after the addition of these solutions were expected as they are what help us extract the DNA plasmid for an end product.(b) Agarose gel electrophoresisAfter placing the DNA plasmid in the wells electrophoresis was carried out. The results were then obtained and recorded.The size of the DNA fragment is immovable from its electrophoretic mobility. The DNA fragments of know molecular weight markers are run on the gel and a graph of log MW against migration distance is drawn.There are tether different forms of agarose DNA first theres the devote circular plasmid DNA this is the first band that occurs on th e picture. The circular plasmid is adouble-strandedcircularDNAmoleculethat has been nicked in one of the strands to allow the release of any super-helical turns present in themolecule. The blossom circular plasmid migrates more slowly than a linear or super-coiledmoleculeof the same size this is due to associated differences inconformation, or shape, of themolecules. this is why it is the first band that occurs on the picture result.Linear DNA has free ends, either because both strands have been cut, or because the DNA was linearin vivo. The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments ( all over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules resperate, with the bulk of the molecule following the leash end through the gel matrix.Restrictio n digestsare frequently used to analyse purified plasmids. These enzymes specifically go to pieces the DNA at certain short sequences. The resulting linear fragments form bands aftergel electrophoresis. It is possible to purge certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments. This is neither fast nor slow in comparison to the other DNA plasmid.The super-coiled Plasmid DNA normally occurs naturally, there is super-coiling in DNA only if there is a replication of a DNA plasmid and this occurs for a small space of time and that is removed by cutting the DNA by specific enzymes, this is part of DNA replication mechinary. This type of DNA plasmid is the fastest as it is the last band shown out of the three this is Because of its tight conformation.The picture above shows the results obtained from the agarose gel electrophoresis. The lane numbers are marked over the wells. The lane before lane 1 that is titled M is the molecular w eight marker.All three forms of plasmid DNA is present in this result, the open circular, the linear and the supercoiled. There is an extra band of RNA present however not clearly visible this is because the RNA fragments migrated ahead of dye front as diffuse a band, the ribonuclease gets rid of this band, a blue tracking dye cause the low smudge under the DNA plasmid and beneath that is the barley visable RNA. RNA is very unstable under these conditions, as a result RNA can be completely degraded befor the ribonuclease has been added.It can be seen that DNA is present more in one band then another, however the one with the less amount could have a bigger fragment. There seems to be logarithmic relationship amongst the size of the DNA fragment and the distance it travels on the gel. A standered curve can be make if we measure the length the bands in different lanes travelled if the fragment sizes are known. The more points plotted and the larger the separation there is on the gel , the results will be more accurate.ConclusionThe experimental procedures carried out were a success, the DNA plasmid was obtained and the agarose gel electrophoresis resulted with in a clear picture as shown and sketch above, of the DNA being successfully separated.The uses of purified plasma in DNA research is for molecular cloning.