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electrophoresis and restriction digestion




electrophoresis and restriction digestion, 3 questions.;Attachment Preview;restrictionenzyme.doc Download Attachment;Appendix C: Use of quantitative DNA ladder 2014;Restriction Digestion and Gel Electrophoresis;R6. Show a figure with the picture of your gel (step 33) and the corresponding labels.;Estimate the migration distance of all bands. Plot logMW (bp) of the MassRuler DNA;ladder (figure 3 and Appendix C) vs. the migration distance (show the figure). Estimate;the MW (bp) of all DNA fragments in your four samples.;(BELOW IS THE GEL, further below is background information and and;procedures, plus appendix);label first well Eco, Hind, E+H, Control, Ladder;Ladder;R7. From the number and size of the fragments observed in your three digested;samples, create a map of the plasmid showing the relative location of the restriction;sites for EcoRI and HindIII.;R8. Looking at your gel electrophoresis identify the format(s) your the undigested;plasmid has adopted? Were your digestions (EcoRI and HindIII) complete? Why or;why not?;F. Plasmid;Appendix C: Use of quantitative DNA ladder 2014;Plasmids are circular, double-stranded DNA molecules that exist independently of;chromosomal DNA in bacterial cells and can range in size from 1-200 kilobases. These;plasmids can be stably inherited by the daughter cells following replication or;transferred to other bacteria by a process known as horizontal gene transfer (HGT). A;common form of HGT is called conjugation, where bacteria can transfer plasmids by;direct cell-to-cell contact. Most naturally occurring plasmids carry genes that provide an;advantage to its host, such as antibiotic resistance or the production of specific;enzymes.;Scientists have taken advantage of plasmids in order to manipulate genes. These;experimental plasmids, also referred to as vectors, have undergone a variety of;modifications in order to make them more useful to scientists. They can be used to;clone and transfer genes, as well as for the expression of recombinant proteins. In the;laboratory, plasmid transfer is achieved by temporarily rendering cells permeable to;small DNA molecules, using chemicals (such as calcium chloride, a source of divalent;cations) or electrical shock. Plasmids under relaxed replicative control may be present;in anywhere from 10 to over 500 copies/cell, thus allowing the host to produce a large;amount of plasmid DNA from a given number of bacterial cells, which is especially;useful for cloning purposes.;The types of plasmids used in the lab usually contain at least the following features;(figure 2);i) Origin of replication: Allows the plasmid to replicate independently of the;bacterial cells chromosomal DNA. This is the DNA sequence that will be;recognized and bound by the replication machinery.;ii) Multiple cloning site (MCS): A short stretch of DNA that contains recognition;sequences for several different restriction enzymes. This allows us to insert;fragments DNA, such as our genes of interest, into the plasmids. It is important;to note that the restriction enzyme recognition sequences found within the MCS;are unique and are not found elsewhere on that same plasmid. When the;plasmid is replicated, so is the inserted DNA, allowing for amplification of this;newly introduced sequence.;Appendix C: Use of quantitative DNA ladder 2014;iii) Selectable marker: It is impossible to visually tell the difference between a;bacterial cell that carries a plasmid and one that does not. That is why plasmids;used in the lab have been engineered to contain a selectable marker, a gene that;imparts a certain trait to the bacteria and allows us to tell whether the cells;contain our plasmid of interest. A gene that confers antibiotic resistance;(ampicillin-resistance is commonly used) is often found on these types of;plasmids. Only the bacteria that contain the plasmid will express the antibioticresistance gene, allowing them to survive and replicate in an environment;containing ampicillin. Bacteria that do not contain the plasmid will not survive.;Figure 2. Plasmid DNA. Schematic;of a generic plasmid depicting;their common features: an origin of replication, the multiple cloning site (MCS) with a;density of unique recognition sites for restriction endonucleases and a gene conferring;antibiotic resistance, the most commonly used selectable marker.;G. Agarose Gel Electrophoresis of DNA;Gel electrophoresis is a technique used to separate macromolecules - especially;proteins and nucleic acids - that differ in size, charge or conformation (6, 7). When;charged molecules are placed in an electric field, they migrate toward either the positive;(anode) or negative (cathode) electrode according to their charge. In contrast to;proteins, which can have either a net positive or net negative charge, nucleic acids have;at neutral pH a negative charge, due to the phosphate groups of their backbone. The;relative migration distance of each molecule is determined by the charge density of the;molecule and the resistance of the matrix (or gel) media to the passage of the molecule.;In DNA/RNA agarose electrophoresis (8), a gel of agarose is cast in the shape of a;horizontal thin slab, with wells for loading the sample close to the cathode (usually;connected to the black wire). The gel is immersed within an electrophoresis buffer that;Appendix C: Use of quantitative DNA ladder 2014;provides ions to carry a current and some type of buffer to maintain the pH at a;relatively constant value.;Agarose;is;a;polysaccharide;extracted from seaweed. Agarose gels;are prepared by mixing agarose powder;with buffer solution to a final;concentration of 0.5 to 2%, followed by;heating until a clear solution is obtained.;Most commonly, ethidium bromide (final;concentration 0.5 g/ml) is added to the;gel at this point to facilitate visualization;of DNA after electrophoresis. However;as ethidium bromide is a toxic mutagen;we will use a safer DNA stain instead;known as SYBR safe (Invitrogen). After;cooling the solution to about 55C, it is;poured into a casting tray containing a;sample comb and allowed to solidify at;room temperature or in the cold. The;porosity of the gel is inversely related to;the agarose concentration. By varying;the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be;separated. The higher the agarose concentration, the "stiffer" the gel and the smaller;the size of the DNA or RNA fragments that can be separated.;Following separation, DNA fragments are visualized by staining with SYBR-safe.;This fluorescent dye intercalates between bases of DNA and RNA. It is often;incorporated into the gel so that staining occurs during electrophoresis, but the gel can;also be stained after electrophoresis by soaking in a dilute solution of SYBR-safe. DNA;or RNA bands appears in red-orange color when the gel is exposed to UV light.;Fragments of linear DNA migrate through agarose gels with a mobility that is;inversely proportional to the log of their molecular weight. Circular forms of DNA;migrate in agarose differently from linear DNAs of the same mass. Several factors have;important effects on the mobility of DNA fragments in agarose gels, and can be used to;advantage in optimizing separation of DNA fragments. Among these factors are;agarose concentration, voltage (as the voltage applied to a gel is increased, larger;fragments migrate proportionally faster than small fragments), electrophoresis buffer;and SYBR-safe (when present in the gel). The molecular weight of a linear DNA sample;can be determined by running a mixture of linear DNA fragments of known size under;the same conditions (Figure 3).;Figure 3: MassRuler Express Forward DNA ladder marker. The MassRuler Express;Forward DNA ladder is constituted of 12 DNA fragment varying in size from 100 base;pairs (bp) to 10,000 bp. Predetermined quantity of each fragment were mixed to;Appendix C: Use of quantitative DNA ladder 2014;produce this marker. These quantities are dependent on the volume of marker to be;loaded on the agarose gel. For example, 5 microlitres (L) of the marker contains 50 ng;of the 1000 bp fragment whereas 10 L of the same marker will contains 100ng of the;same fragment. It is important to remember that linear DNA fragments migrate through;agarose gels with a mobility that is inversely proportional to the log of their;molecular weight;H. Restriction Endonuclease Mapping of a Plasmid;Restriction endonucleases are a family of site-specific endonucleases that cleave;the phosphodiester bond in the phosphate back bone of DNA. This cleavage is a;hydrolysis reaction, adding a molecule of water across the phosphodiester bond;breaking it and generating a 5 phosphate and a 3 hydroxyl end (Figure 4A). These;enzymes recognize short (4-8bp), often palindromic (identical when read 5 to 3 on;each strand) sequences. The cleavage of both strands of the DNA backbone generates;either blunt or sticky ends, depending on the specific enzyme used (Figure 4B). These;ends are exploited for many molecular biology used to manipulate DNA, most notably;cloning.;When plasmid DNA is isolated and run on an agarose gel, several bands may be;seen. The predominant form a plasmid takes is a supercoil, often likened to an over;twisted telephone cord, the double helix twists on itself yielding a compact piece of DNA;that migrates faster than its linear form. A plasmid may be damaged or in the process;of being replicated at the time of isolation resulting in a nicked formation, likened to a;big floppy circle, being adopted. The nicked format migrates slowly mimicking a DNA;fragment longer than its linear form. Finally, during alkaline lysis it is possible to use;overly harsh conditions resulting in a permanently denatured intact single strand of the;plasmid, this form migrates the fastest on an agarose gel (Figure 4C). Following a;restriction digest it is often helpful to run both a digested aliquot and an undigested;control on an agarose gel to confirm that the digestion is complete based on the;banding pattern observed.;With each restriction enzyme having a unique recognition sequence, each time an;identical piece of DNA is digested with the same enzyme, a characteristic fragmentation;pattern will be observed. Exploiting this reproducibility, restriction mapping consists of;digesting DNA with a series of restriction enzymes and separating them on an agarose;gel to visualize the resulting fragments (Figure 4C). These fragmentation patterns are;then be used to characterize the relative location of the restriction sites be it on an;unknown piece of DNA, as will be done in this experiment or to track and confirm the;results of manipulating DNA (such as when inserting of a gene of interest in to a;plasmid).;Appendix C: Use of quantitative DNA ladder 2014;Figure 4. Restriction digestion and plasmid mapping. A) Adding a molecule of;water across the phosphodiester bond breaks the phosphate backbone of the DNA;generating a 5 phosphate and a 3 hydroxyl end on the resultant DNA fragments (figure;modified from http://chem B) Digestion with restriction enzymes may;result in sticky ends, as seen for HindIII and EcoRI or blunt ends, as seen with EcoRV.;C) A schematic depicting the restriction digest of a plasmid using two enzymes: where a;single digest with each enzyme and a double digest were completed and compared with;the undigested plasmid, in all its possible conformations and a molecular weight;marker.;Appendix C: Use of quantitative DNA ladder 2014;Appendix C: Use of quantitative DNA ladder 2014;PROCEDURES;In experiment 1 you will extract the E. coli DNA. For experiment 2, a stock solution;of E. coli DNA from a commercial source will be provided by the lab technician so that;you can start the first melting curves (step 16) early in the lab session. In this;experiment, you will analyze the effect of salt and a denaturing agent on DNA structure.;Using the same melting technique, you will compare the homogeneity and integrity of;the in-lab prepared DNA with a commercial source of E. coli DNA. In experiments 3 and;4 you will be introduced to the techniques of restriction digestion and DNA;electrophoresis on agarose.;Before performing these experiments, you should watch the following videos;Gel electrophoresis Part 1 (13:05) or (;v=3ukaT_Ih9d8);Gel electrophoresis Part 2 (8:13) or (;v=_QxxB65Gi78);EXPERIMENT #1: Isolation and characterization of bacterial DNA;a)DNA extraction;1. You are provided with 60 mg of Escherichia coli, strain B, (a gram negative;bacterium) suspended in 4 mL of cold Standard saline-EDTA (0.15 M NaCl in 0.1;M ethylene-diamine tetra-acetate EDTA, pH 8.0) in a 50 mL Falcon tube.;2. Add 375 L of 25% sodium dodecyl sulfate (SDS) and mix gently by inversion.;(SDS is a detergent and will foam extensively, you could also easily shear the;DNA).;CAUTION! The SDS solution may irritate the skin and eyes. Use gloves and;safety glasses.;(;3. Incubate the mixture in a 60C water bath for 10 minutes and then cool to room;temperature.;Appendix C: Use of quantitative DNA ladder 2014;4. Add 0.725 mL of 6.0 M NaClO4 and mix gently.;CAUTION! The perchlorate solution may irritate the skin and eyes. Use gloves;and safety glasses. (;5. Add 5.0 mL of chloroform:isoamyl alcohol (24:1, v/v) in the fume hood.;CAUTION! Chloroform and isoamyl alcohol are toxic and irritate skin, eyes and;respiratory tract. Keep away from sparks and flame. Use gloves and safety;glasses. Avoid inhalation. Work in the fume hood.;(;(;6. Mix gently on a waver for 5 minutes.;7. Centrifuge at 12,000 xg for 5 minutes. (Tubes should be balanced!).;8. Carefully remove 70% of the upper aqueous phase by aspirating the liquid off;with a plastic pipette and transfer it to a 15 mL Falcon tube. Avoid contaminating;your sample with the denatured protein that collects at the interface between the;aqueous and organic phases. Dispose of the chloroform phase in the appropriate;waste container.;9. Gently layer 10 mL of 70% ethanol over the aqueous phase in the tube. Seal the;tube with its cap and mix gently and continuously until the ethanol becomes fully;mixed with the aqueous phase (Observe the precipitation of the DNA).;10. Retrieve the DNA using a glass rod and immerse it into a microtube containing 1;mL of 70% EtOH. Mix it gently in this solution to remove the salts.;Appendix C: Use of quantitative DNA ladder 2014;11. Squeeze out as much liquid as possible from the spooled mass by lightly;pressing the stirring rod against the side of the tube. Place the glass rod in an;upright position and allow it to drain for at least 10 min. Failure to remove the;alcohol effectively will lead to difficulties in dissolving the DNA in the next step.;Further removal of ethanol can be achieved with a stream of air.;12. Dissolve a fraction of the crude DNA on the glass rod by stirring the DNA into 2;mL of 15 mM citrate buffer (sonicated), pH 7.0 in a 15 mL centrifuge tube. Gently;swirl the glass rod back and forth until the DNA becomes detached from the rod.;Seal the tube and place it on a waver for 5 min.;13. Transfer 1 mL to a 1.5 mL microtube and centrifuge it for 1 min at 13,000 rpm.;b) DNA characterization;You will determine your DNA yield as well as the degree of purity and integrity of a;DNA sample prepared as above.;14. Pipette 200 L of your dissolved DNA from step 13 into a quartz cuvette;containing 800L of 15 mM citrate buffer. Mix by inversion. Read and record the;absorbance at 234, 260 and 280 nm of your sample with the UVspectrophotometer, using the 15 mM citrate buffer as the blank.;15. Prepare a dilution of your DNA sample with a final volume of 1mL in 15 mM;citrate buffer, pH 7.0 in order to have an A260 between 0.4 and 0.5. Calculate and;record your dilution factor. This solution will be used for the DNA melting curve;experiment (step 18).;EXPERIMENT #2: DNA melting curves;For a melting experiment you will follow the change in absorbance at 260 nm of;the DNA in function of the temperature. You will use the Cary100 Bio system that can;analyse 12 DNA samples simultaneously.;Melting experiments will be performed on the following DNA samples;Appendix C: Use of quantitative DNA ladder 2014;a) E. coli DNA from a commercial source;b) E. coli DNA from a commercial source, in the presence of salt;c) E. coli DNA from a commercial source, in the presence of dimethyl;formamide;d) E. coli DNA from step 15.;Each team will perform one of the three meltings a-c plus its own sample (d).;16. Prepare 1 mL of a 1:20 dilution from the stock commercial DNA solution as;indicated below: (Take 50 L of the stock solution and add it to 950 L of the;corresponding buffer).;Note: All buffers have been de-aerated by sonication. This will prevent bubble;formation during the heating process.;solution a: commercial E. coli DNA in 15 mM citrate buffer, pH 7;(Teams 1, 2, 3, 8, 9, 13, 14, 15, 17, 19, 20 and 21);solution b: commercial E. coli DNA in 15 mM citrate, 0.10M NaCl;(Teams 4, 5, 10, 11, 22 and 23);solution c: commercial E. coli DNA in 15 mM citrate, pH 7, 25% DMF;(Teams 6, 7, 12, 16,18 and 24);CAUTION! Dimethyl formamide (DMF) solutions may irritate skin and eyes. If;absorbed, it may harm the unborn child (teratogen). Use gloves and safety;glasses.;(;17. Once samples from all teams are loaded into the Cary 100Bio spectrophotomer;(as directed by your TA), the technician will start the melting experiment.;Appendix C: Use of quantitative DNA ladder 2014;18. Once experiment 1 is completed, each team will set up a second melting;experiment with your own extracted DNA sample. Samples from teams 1 to 12;will be run in one apparatus and samples from teams 13 to 24 in the second;apparatus. Please record the cell number in which your sample is loaded.;19. Once the melting is finished, you will see the two plots of the absorbance and the;1st derivative on the same graphic. The out-put from the instrument (BTM file) will;be saved and posted on the courses web site in the Virtual Campus as a CSV;file. (Excel can open this file and save it as a XLS file which is the Excel format).;For convenience, the files will be named in the following format: L5S12(112)abc.csv or L5S12(1-12)d.csv, where S12 is the second Thursday afternoon;section and (1-12) indicate samples from teams 1 to 12. For your report you will;need the results corresponding to your sample plus any set of the three;commercial samples in different conditions (control, salt and DMF) that were;analyzed the same day. You are to incorporate the four graphics (a - d) in your;report showing the two plots for the absorbance and the 1 st derivative.;EXPERIMENT #3: Restriction Endonuclease Mapping of a Plasmid;In this experiment, an unknown plasmid (100 ng/L) will be digested with a;combination of two different restriction enzymes: EcoRI (10 U/L) and HindIII (10 U/L).;You will be supplied with H2O, a solution of 10X digestion buffer (Buffer O) and an;unknown plasmid (100 ng/L). Each team will be digesting a different plasmid.;20. Prepare the following reaction mixtures in 4 labelled 0.5 mL microcentrifuge;tubes by adding the following reagents in the order they are listed. Ask your TA;for the EcoRI and HindIII restriction enzymes, adding them to the mixture last;rinse the pipette tip with the solution at least 5 times. Always keep your tubes on;ice until loading it into the waterbath.;Appendix C: Use of quantitative DNA ladder 2014;Table 1. Plasmid digestion mixture;Reagents;EcoRI;HindIII;H2O (brown tube);10X Buffer (blue tube);Unknown plasmid (100;ng/L);(Orange tube);EcoRI (10 U/L) (see TA);HindIII (10 U/L) (see TA);Total volume;11 L;2 L;11 L;2 L;EcoRI +;HindIII;10 L;2 L;6 L;6 L;6 L;6 L;1 L;---20 L;---1 L;20 L;1 L;1 L;20 L;------20 L;Control;12 L;2 L;21. Mix the solutions by tapping the bottom of the tubes with your finger. To bring all;the reagents to the bottom of the tube, centrifuge for 15 seconds. (Make sure;your tubes are balanced!);22. Incubate your tubes in the 370C water bath for 1 hour.;23. Once the digestion is completed, add 2.2 L of 10X DNA-electrophoresis loading;buffer to each sample. Mix thoroughly.;Proceed now with electrophoresis on the provided gel (step 29).;EXPERIMENT #4: Agarose gel electrophoresis;N.B. To avoid waiting time, a gel for every four groups is provided. Your TA will;demonstrate how to prepare a gel (steps 24-28). Groups of 4 teams will load a gel;and perform the electrophoresis as described in steps 29-34. Before loading the;sample, you can practice with loading buffer and the demo gel.;24. In a 250 mL bottle, prepare 100 ml of a 1% (w/v) agarose solution in TAE (40 mM;tris-acetate, 1.0 mM EDTA) buffer. Check calculation with your TA before;proceeding.;25. Heat the agarose suspension in a microwave oven. To prevent pressure from;building up inside the bottle, loosen the cap before heating. Use high setting 3 x;1 min, mixing after each minute. Make sure agarose is completely melted.;Appendix C: Use of quantitative DNA ladder 2014;26. Once dissolved, allow to cool to 50-55C (agarose solidifies at about 40C), any;hotter than 55C will melt the gel casting mold.;27. When the gel solution is at 50-55C, add 10 L of SyBr Safe DNA Stain.;28. Place the gel running tray in the gel casting mold and pour the cooled (50-55C);agarose solution into the casting mold. Pour slowly to avoid making bubbles.;Place a 20 well comb at 1 cm from one end of the casting tray. Dislodge any;bubbles with a pipet tip. Allow the gel to solidify for at least 20 min.;29. Remove the comb by slowly lifting one end, making sure not to suck the agarose;out of the bottom of the well.;30. Lift the gel tray from the casting mold and place in the electrophoresis chamber.;Pour in the TAE buffer until the gel is covered by 4-8 mm of buffer.;31. Load 10 L of provided MassRuler Express Forward DNA ladder marker (M) and;15 L of the samples from step 23 in wells 1 to 20 as described in the template;bellow. Place the end of the tip below the surface of the running buffer;immediately above the desired well. Expel the sample slowly (taking care not to;allow any air bubbles to form under the tip), the high concentration of glycerol in;the loading buffer will cause the entire sample to sink to the bottom of the well.;Gel loading template;Appendix C: Use of quantitative DNA ladder 2014;32. Place the lid on the electrophoresis unit and connect the electrodes such that;migration proceeds towards the RED (positive, anode) electrode. Connect the;leads to the power supply and apply 100 V for 45 min.;33. Turn the power supply off, disconnect both ends of the electrodes and remove;the gel tray. (WEAR GLOVES);34. Place the gel and running tray on the UV Gel Doc System and take a picture.;(Ask your TA for help). For convenience, name the file in a similar way as for the;melting data by giving the lab number, the section and the number of the teams;with samples on the gel (e.g. L5S2(5-8)).;Appendix C: Use of quantitative DNA ladder 2014;RESULTS AND DISCUSSION;Appendix C: Use of quantitative DNA ladder 2014;Quantitative DNA Ladder and estimation of length and amount of DNA bands;A quantitative DNA ladder contains known amounts of DNA molecules of different;lengths. It serves as a reference to estimate the size and amount of an unknown DNA;band. A quantitative DNA ladder must be used each time you are loading sample on;and agarose gel, it must be loaded at the same time as your samples.;Length of an unknown;One of the first information you can draw from the use of a DNA ladder is the;estimation of the length of an unknown DNA band. By quickly comparing the length of;your unknown band to the bands of the molecular ladder, you can estimate its length;(Figure 1A). If you want to be more precise, one can a plot describing the log(length) vs;mobility for the different bands of a marker, and then use the equation derived from the;trendline to predict the length of an unknown. Here is how you can achieve that;a) Measure the travelling distance of each band of the marker by using a ruler (from;the well to the middle of each DNA band). See Figure 1B.;b) Plot the log of the length of the DNA fragments against their travelling distances;(Scatter plot in Microsoft Excel). See Figure 1C.;c) Create a linear regression line (Linear trendline option in Excel and check the;Display Equation on chart option. See Figure 1C.;d) You can now measure the travelling distance of your unknown and input this;value in the linear regression equation to get a prediction of the length of your;unknown sample.;As you can see in the presented example, high molecular weight fragments;sometimes affect the results of the linear regression. Large DNA molecules (10,000bp;+) have a really hard time migrating through the molecular mesh of a 1% agarose gel;and therefore, it becomes more difficult to measure the migration distance of these;fragments with precision. If you notice outlier values on your linear regression line, you;can eliminate some of them to improve your R 2 as I did for the example presented.;Amount of an unknown;You can also quantify (estimate) the amount of DNA that was loaded on gel by;direct comparison to the DNA ladder. The intensity of a band is proportional to the;number of SYBR Safe molecules bound onto the DNA and therefore, this intensity will;vary with the number of bp and the amount of the DNA fragment that was loaded on gel.;There are two rules to this type of estimation, first, always try to use a band of the;marker that is closely related in size to your unknown since larger fragments will;intercalate more dye than small fragments, which in turn, will give them greater band;intensity. Secondly, once you have selected a band of the marker for your comparison;use the information given by the manufacturer (amounts of DNA by loading volume) to;know how much DNA is present in that band. Knowing the amount of DNA present in;Appendix C: Use of quantitative DNA ladder 2014;the marker band (from the manufacturer information sheet), you can then apply the;intensity ratio to this number. For example, if you look at gel presented in Figure 1A;the unknown band is 3 times more intense that the 3000 bp marker band. If 30 ng of this;DNA fragment was loaded on gel (10 L of the marker), I can say that I have loaded;~90 ng of my unknown.;Figure 1. Quantitative DNA Ladder and estimation of length and amount of DNA;bands. A) Estimation of a DNA fragments length using the MassRuler Express Forward;marker (Fermentas). DNA quantity presented on the right side of the marker description;sheet correspond to amounts of DNA loaded for each fragment of the marker in relation;to the volume of marker used (top line). B) Determination of the DNA fragments length;by plotting the logarithm of the length of the DNA fragments of the marker against their;migration distances. C) Linear regression of the log (fragments length) against the;Appendix C: Use of quantitative DNA ladder 2014;migration distance. Points located at the end of the regression line were eliminated to;improve the R2 value of the line.;Appendix C: Use of quantitative DNA ladder 2014;Appendix D: Supplementary figures 2014;Appendix D: Supplementary figures 2014;Lab 3 Supplementary Figure 1. Enzyme inhibition effects observed using;Lineweaver-Burk (LWB) representation. A) Michaelis-Menten plot of v0 against;increasing [S], where vo approaches Vmax at high [S] and half of this maximum velocity;corresponds to KM. B) Taking the reciprocal of the Michaelis-Menten equation and;plotting 1/v against 1/[S] allows for analysis by linear regression, where the slope =;KM/Vmax, y-intercept = 1/Vmax and x-intercept = -1/KM. In the case of COMPETITIVE;inhibition, Vmax is unaffected but the amount of substrate needed to achieve it increases.;This is graphically visible by a change in the shape of the curve for the MichaelisMenten plot due the increase in Km by factor of, where: = 1 + [I]/KI (C) and an;increase in the slope ((Km/Vmax) of the LWB plot along with an increased x-intercept (1/Km) (D). In the case of UNCOMPETITIVE inhibition the both the Km and Vmax are;decreased by a factor of, where = 1 + [I]/KI. This results in an overall lowering of;the Michaelis-Menten curve (E) and an upward shift of the LWB plot. While the slope of;the LWB remains unchanged, the y-intercept (/Vmax) increases and the x-intercept;decreases ((-/Km) (F). Finally in the case of mixed inhibition the V max decreases;(Vmax /) while the Km increases (Km/This is observed as a lowering and flattening;of the Michaelis-Menten plot (G) and an increase in the slope (Km/Vmax) of the LWB plot;due to the increased y-intercept (/V max) and decreasing x-intercept (-/Km)(H).;Lab 3 Supplementary Figure 2. Enzyme inhibition effects observed using the;Hanes representation. A) Michaelis-Menten plot of v0 versus increasing [S], where vo;approaches Vmax at high [S] and half of this maximum velocity corresponds to K M. B);The Hanes plot is an alternative linear model that can be obtained by multiplying the;Lineweaver-Burk equation by [S] and plotting [S]/v against [S]. In this linear-regression;the slope corresponds to 1/V max, y-intercept to KM/Vmax and x-intercept to -KM. In the case;of COMPETITIVE inhibition, Vmax is unaffected but the amount of substrate needed to;achieve it increases. This is graphically visible by a change in the shape of the curve;for the Michaelis-Menten plot due the increase in K m by factor of, where: = 1 + [I]/KI;(C). The slope of the Hanes plot remains unchanged, while the y-intercept (Km/Vmax);increases and the x-intercept decreases (-Km). (D). In the case of UNCOMPETITIVE;inhibition the both the Km and Vmax are decreased by a factor of, where = 1 + [I]/KI.;This results in an overall lowering of the curve in the case of the Michaelis-Menten plot;(E) an increase in the slope (/Vmax) of the Hanes plot due to an increased x-intercept (Km/) (F). Finally in the case of mixed inhibition the V max decreases (Vmax /) while the;Km increases (Km/This is ovserved as a lowering and flattening of the MichaelisMenten plot (G) and an increase in the slope (/Vmax) of the Hanes plot due to the;increased y-intercept (Km/Vmax) and decreasing x-intercept (-Km/)(H).;Appendix D: Supplementary figures 2014;electrophoresis and restriction digestion


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