Isolation of DNA is a very important technique which is thefoundation for many types of techniques such as the diagnosis of many geneticdiseases as well as fingerprinting DNA. How much amount and purity required,the DNA type is what makes the difference for the different methods for DNAisolation. There were three different E.
coli cultures the aim was the analysethe DNA of the E. coli. Multiple amountsof techniques were used to manipulate and isolate the DNA from E.
coli. Westart of by Isolating the plasmid DNA from the 3 cultures using alkaline lysis.Alkaline lysis is an extraction method used to isolate plasmid DNA frombacteria. Next the DNA which has beenisolated restriction enzymes is used for digestion. Restriction enzymes areable to cleave DNA and make them into fragments and this is within the moleculeat sites called restriction sites. Bacterial transformation was also done. Finally,is the analysis of the E.
coli which is transformed and this is achieved byusing agarose gel electrophoresis.MethodPractical 1Three E. coli cultures A, B and C were provided which hadbeen grown overnight shaken at 37°C. The bacterial pellet is dissolvedin 100 ml of solution 1.Solution 1 contains 50Mm glucose the glucose aims to provide an osmotic balancebetween the cell and the solution and this prevents the cells from bursting atthis stage. Solution 1 also contains 25Mm Tris (pH 8.
0) this is used tostabilise the ph in the solution. EDTA10Mm is also a chemical which is necessary to allow DNA degrading enzymes. Themain purpose for EDTA is to bind to magnesium and calcium and this stops theDNA from degrading. The EDTA is also able to stabilise the DNA phosphatebackbone as well as the cell wall.Next solution II is added, solution II is 0.
2M NaOH and 1% (w/v)SDS. This strong alkaline solution is able to disturb the cell membranes andallows to come in contact and denature the plasmid and chromosomal DNA. Thecell contents made contact with the extracellular chemicals which allowed EDTAto chelate with the metal ions in the cells. SDS precipitate with proteins inthe cell contents and form insoluble complex. As a result, precipitates wereobserved in the solution.
Chromosomal DNA and plasmid DNA were denatured by thehigh pH in the solution. The process is known as denaturation as it Solution 3 is added next which is 3M potassium acetate pH4.8. The potassium acetate is able to decrease the alkalinity of the solutionso it able to renature the plasmid DNA but does not renature the chromosomalDNA. The ssDNA can re nature the dsDNA because the hydrogen bonds between thesingle stranded DNA is re-established.
Through hydrophobic relations a whiteprecipitate is formed by the SDS, denatured cellular proteins and the singlestranded genomic DNA all sticking together whereas the double stranded plasmiddissolves in the solution.At this point most of the cells debris is separated from theplasmid DNA but in the solution there is the debris the salts, Rnase and EDTAso the solution has to be cleaned up and the plasmid DNA concentrated. 70%ethanol is added next and it is able to change the DNA’s structure as theyaggregate and precipitate from the solution. Using centrifugation, the DNAwhich is precipitated can be separated. Practical 2From the E. coli which had been isolated next we begin todegrade the DNA using restriction nucleases.
Restriction enzymes cut DNAmolecules in specific areas to cut them into smaller fragments. Different kindof DNA sequences are cut and recognised by different restriction enzymes. Theserestriction enzymes also need a buffer which is suitable this includesmagnesium as a co factor.
A certain concentration and a Tris to buffer the ph.For different kinds of enzymes there are different optimum salt concentrations.Samples B and C are isolated with 10 units of enzyme. There are 2 tubes calledtube BR and tube CR. Tube BR contains DNA B, 10 x EcoR1 buffer, EcoR1 enzymeand water, Tube CR contains the same but instead of DNA B it contains DNA C.There is a specific order in which these are added. Firstly, water is addedthen it is the buffer, the DNA and then finally it is the enzyme.
The reasonfor this order is because a suitable environment has to be created before theenzyme is added. Eco R1 is basically a restriction enzyme isolated from E.coli, which at particular locations cuts DNA double helix at specificrestriction site.
EcoR1 is able to make cuts in the backbone of both of thestrands, and this allows there to be two sticky ends at the cutting site of theDNA. There is a specific sequence whichthe EcoR1 can recognise this sequence is GAATTC and the enzyme cuts in betweenthe G and A on the strand which is complimentary. To start the solution isadded orderly with water, buffer and the enzyme. The water is used to dilutethe buffer because the manufacturer concentrates the buffer. The EcoR1 bufferis there as it is the optimal buffer used for the enzyme performance. When theconditions are finally suitable for the enzyme it is added.
This is when itopens up or fragmentise the plasmid DNA. Once the both the tubes had beencompleted they were incubated. Transformation of plasmid Now a technique known as bacterial transformation is usedand two tubes B and B are diluted in a Tris buffer at pH 7.6 and this makes up40 folds of the final volume of the mixture, as they have been diluted they arelabelled diluted B and diluted BR.
The purpose of bacterial transformation isto introduce DNA into bacterial cells. There are many techniques used toachieve this but the reliable technique is a heat shock technique. When the DNAhas been taken up it has to either join with a host genome or autonomouslyreplicate. Circular forms of DNA are the only DNA which are going to be able toreplicate, the linear form which use restriction enzymes will not be able totransform. The circular form when introduced to E. coli will be able toreplicate. The heat shock technique uses calcium chloride which createsa calcium rich environment, between the plasmid DNA and the bacterial cellularmembrane the rich calcium environment cancels the electrostatic repulsionbetween them. In the bacteria pores are created as there was been a suddenincrease in the temperature so this allows the entry of plasmid DNA to thebacterial cell.
When the cell takes upthe DNA it establishes itself to create a steady tranformant. In the practicalthe unknown strain of E.coli cells were added with calcium chloride and precooled in ice. The procedure is repeated twice and kept in ice. At the sametime tube 1 which contains no plasmid DNA is prepared, tube 2 containingdiluted plasmid B is prepared and tube 3 containing diluted plasmid BR isprepared. The pre cold competent cells were added to the tubes 1 2 and 3 andmixed gently. As the cells are fragile, it is important to avoid using thevortex.
After 15 minutes the tubes were shocked with hot water at 42°C. At thisstage the cell membrane becomes thinner and plasmid DNA can enter the cellbody. After 2 minutes the tubes were set in ice for 5 minutes to allow the cellmembrane to recover. L broth is added to each tube and water bathed at 37°C forat least 20 minutes. After that cells in each tube were transferred LB ampplate spread and incubated overnight.
Practical 3Agarose Gel electrophoresis is a common used method foranalysing the size, purity, quantity and the sequence of DNA molecules andplasmid DNA molecules. Agarose is a polysaccharide it is a one of thecomponents to agar and is extracted from red seaweed. It is also made up of anhydrous-galactoseunits.
There are many reasons why the agarose gel is beneficial for gelelectrophoresis. Between the polysaccharide unit’s non-covalent bonds areformed by the agarose gel. A sol state is formed as non-covalent bonds hold thestructure of the agarose gel so it undergoes a phase transition at high temperature,When the running buffer and the agarose powder is mixed it creates the gel withlater arrangement of the sol state at a higher temperature and also arrangedcooling.To start an agarose gel is prepared by the molten agarosebeing poured in the former. Wells are formed in the DNA sample for the DNA tobe loaded by combs, they are then left for approximately 20 minutes to set.Once the gel has been set TBE buffer is used to carry a current and provideions it is also able to maintain the Ph. As we know DNA is negatively charged, so when the electricfield is applied for the period of electrophoresis there will be movement ofthe DNA towards the anode which is the positive pole.
The sample loading wellsbe towards the negative pole which is the cathode, so when the gel is placed inthe electrophoresis tank it is orientated. A loading buffer solution is used to treat the plasmidsample before it is loaded on the gel. The density is increased of the sample as the loading buffer containsglycerol.
DNA is able to travel towardsthe positive electrode as the larger fragments are slowed down in compare tothe smaller fragments which is why they do not travel far. A band is alsoformed as all the fragments gather at a point and travel at more or less thesame rate. So now when all the fragments have travelled and separated the differentsized fragments, there is a dye known as SYBER-SAFE and this is used tovisualise the DNA. When the dye is hitby a UV radiation there is an orange coloured fluorescent light. Finally, the UV trans illuminationphotographs the gel which contains the stained DNA molecules.
A loading buffer completes the circuit as wellas balances the pH in the gel.ResultsThe transformed E. coli from tube 1, 2 and 3 were grown inthe agar plate. Tube contained 0 colonies, tube 2 contained 300 colonies andtube 3 had contained 5 colonies. Tube 1 contained 0 colonies as it onlycontained buffer.
Tube 2 had contained 300 colonies as it contained circularplasmid DNA. Tube 3 had only contained 5 colonies as it only contained linearDNA so the only way it may have contained colonies may have been contaminationor mutation. From the results Tube C shows it did not travel far as themolecules were large or had a low molecular charge so they were not able passthrough the gel network. Incomplete precipitation of the chromosomal DNA couldbe a possible error.
It is known that tube A contains no plasmid but tubes B andC contain plasmid. It is because the results show that there are 2 DNA bands onboth tubes B and C but there are no DNA bands in tube A. The plasmid DNA whichis within E. coli is in a circular form. The bands on the top of tubes B and C are known as nick DNA they werelinearized by alkaline solutions and are not able to renature. Circular DNA isable to travel further as its shape causes less friction and it is smaller insize. The nick DNA does not travel far as it is in a linear form which causesmore friction. And it has a larger molecular size.
Plasmid DNA in tube C is cleaved into two fragments by therestriction enzyme because it contains two recognition sequences. The band ofthe two fragments can travel further than the band of plasmid DNA C becausethey experience less resistance. Therefore, the band of the linear plasmid DNAC has lower position than circular plasmid DNA C. Whereas plasmid in tube Bcleaves at only one recognition site the circular plasmid opens up forming anick DNA. Which experience higher friction than the circular plasmid DNA. Therefore,circular plasmid DNA C can travel faster than the nick plasmid DNA C forming aband at a low position.The marker X contains linear double stranded DNA with nomolecular weight. When it is loaded in electrophoresis, the DNA with differentmolecular weight runs to different position and forms a DNA ladder.
Bycomparing the bands of linear plasmid DNA from B and C with the ladder themolecular weight of the plasmids is known. Linear plasmid DNA of E. Coli B has a molecular weight of 3kbp. Thelinear plasmid DNA fragments have molecular weight of 4.7 and 5.
3 kbp thereforethe molecular weight of plasmid DNA C is 10 kbpDiscussion and ConclusionTo start most of the macro molecules such as the proteins,chromosomal DNA and high molecular weight RNA were all removed by the processof Alkaline Lysis. But from the results it has shown there was somemacromolecules which had remained in tube C. The possible reason for the Tube Ccontaining the macromolecules could be Tube B+ had the RNA degraded using theRNase. When the alkaline lysisexperiment was performed there was no single stranded linear DNA whendenaturing was done, and this has shown that there was control on the pH.
5colonies of antibiotic resistant were grown on plate 3 by linearized plasmid B.There was linearized plasmid in tube BR but at low points the reason for thisis there was no circular plasmid DNA seen in electrophoresis. Post inspectionshould be done on the colonies formed on the L-amp plate. When bacterial transformation had been carriedout there may have been a chance some of the plasmid B which is linearized hasmerged with the chromosomal DNA.