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(a) The aim of this experiment was to successfully isolate a DNA plasmid from E.Coli cells (Escherichia coli). We then use commonly performed a method commonly used in biochemistry and molecular biology called agarose gel electrophoresis. This is used to separate DNA and RNA fragments according to length are used to estimate the size and charge of the DNA and RNA fragments or to separate protein by size.
In this procedure as stated above, we used e.coli as these are plasmid containing cells. These cells were placed in a buffer and mixed 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 like chromosomal DNA. Using 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 due to the lyses of the cells which solubilises protein and denatures the DNA.
Subsequent neutralization is potassium acetate allows only covalently closed DNA plasmid DNA to reanneal and stay solubilized. Chromosomal and plasmid DNA precipitate in a complex formed with potassium and SDS which is removed 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 protein
Plasmid DNA is concentrated by from the supernatant by ethanol precipitation. Plasmid DNA isolated by alkaline lyses is suitable for most analyses and cloning procedures without further purification however if the isolated plasmid DNA is 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 backbone. And like any other biological macromolecules can 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 smaller DNA moves faster than the larger molecules of DNA as the smaller ones fit through the whole easier. This causes the DNA to be separated by size and can be seen visually. To make the electrophoresis to function and separate DNA molecules it must contain an electrophoresis chamber.and power 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 can 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 undergone. Ethidium bromide, is a dye used to stain the nucleic acids.Â . Tran illuminatorÂ (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.Â
Method for plasmid isolation
1.5 ml of culture that contains E.coli cells containing the plasmid pUC118 was inserted into an Eppendorf tube.
This was then centrifuged at 13000 rpm for two minutes
The 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 pellet
200 micro-liters of solution A was added to the bacterial pellet. This ensured that the suspension is homogenized (mixtures are well separated
400 micro-liters of solution B was then added and mixed well these solutions contain the SDS and sodium hydroxide. This neutralizes the solutions
300 micro-liters of solution C which contains the potassium acetate which was also mixed and then was incubated on ice for 10 minutes
This mixture was the centrifuged at 13000rpm for 5 minutes
750 micro-liters of this supernatant was transferred to a new Eppendorf tube whilst ensuring none of the precipitate was interfered with
10 micro-liters if RNAse solution was added to a duplicate tube and labeled as R+
450 micro-liters of isopropanol was added to each test tube and mixed well
This was then centrifuged at 13000rpm for 5 minutes
The supernatants were then carefully removed and the DNA was retained
400 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 room temperature
Each pellet was then dissolved in 10 micro-liters of TE buffer
Q1 The viscosity after 400 micro-liters of solution B was added and mixed a low viscosity was observed 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 electrophoresis
The sample obtained from the experimental procedure above were then examined using the method of agarose gel electrophoresis
The RNAse treated and untreated plasmids were examined.
10 micro-liters of loading buffer was added to 10 micro-liters of DNA for each sample
The 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 minutes
It was clear that the current was flowing as bubbles were observed to be coming off the electrodes.
The negatively charged DNA migrated 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 size
(a) Isolation of DNA plasmid
The 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 protect the DNA from DNAses which are degradative enzymes; the EDTA also binds divalent cations that are necessary for DNAse activity. The solution B contains SDS which is a detergent and NaOH. This neutralizes the solution, the alkaline mixture also causes the cells to rupture and the SDS the lipid membrane is broken apart 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. TheÂ E. 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 electrophoresis
After 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 determined 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 three different forms of agarose DNA first there's the open circular plasmid DNA this is the first band that occurs on the picture. The circular plasmid is aÂ double-strandedÂ circularÂ DNAÂ moleculeÂ that has been nicked in one of the strands to allow the release of any super-helical turns present in theÂ molecule. The open circular plasmid migrates more slowly than a linear or super-coiledÂ moleculeÂ of the same size this is due to associated differences inÂ conformation, or shape, of theÂ molecules. 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 linearÂ in 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 (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 leading end through the gel matrix.Â Restriction digestsÂ are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' afterÂ gel electrophoresis. It is possible to purify 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 weight 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 black 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 between the size of the DNA fragment and the distance it travels on the gel. A standered curve can be made 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.
The 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 outlined above, of the DNA being successfully separated.
The uses of purified plasma in DNA research is for molecular cloning.