The total weight of the practicals are

1 Enzyme Kinetics Semester 1 2018 MSE-2025 Molecular Biology and Biochemistry General Info Dates: Mon 22nd October to Wed 24th October inclusive Monday 0900-1300 Brambell B1 Tuesday and Wednesday 0900-1700 Brambell B1 The total weight of the practicals are 50% of the final module mark (25% for Chriss, and 25% for Edgars practicals) Write-up Turnitin submission date: Friday 9th November 2018 (2359 hrs) Please work in pairs where possible the volumes of reagents have been calculated accordingly. Before you start At times, it will also be necessary to wear eye protection, and when handling any reagents or equipment associated with this practical, protective glove should be worn. Hazard and Risk Assessment Several chemicals involved in this practical should be handled with care. Therefore, it is a good idea if students treat ALL the solutions used in this practical with the same degree of care, being sure to wear a buttoned-up lab coat, protective gloves and eye protection when handling these reagents. In addition, Good Laboratory Practice should be observed at all times. 1. NaOH is highly alkaline and causes corrosive burns. 2. Para-nitrophenol is an irritant, and skin contact may cause an allergic response 3. Kanamycin is not dangerous, but can cause a serious allergic reaction in some individuals. You may not know that you are allergic please handle with care. If you are allergic to any antibiotics, please have your lab partner carry out this part of the work. 4. DH5 E.coli bacteria: these are non-pathogenic, and are incapable of causing enteric infections nonetheless, please handle with care. 5. Miniprep kit reagents: P2 buffer is corrosive and contains NaOH and Sodium Dodecyl Sulphate (it busts open bacteria in seconds so it is obviously a harsh chemical). Buffer P3 is a serious irritant. Buffer PB is a flammable irritant. 2 Overview This practical has several component parts: Day 1 We will cover some basic background enzyme kinetics, and information on benzonase and acid phosphatase. Calculations for the next two days a number of blank spaces have been left in the experimental instructions. These calculations will be carried out during the first practical session as there will be sufficient downtime we will confirm the correct answers before continuing with the protocols. Bacterial transformation with a plasmid for use on Day 3 in benzonase assays, and plating of transformants on kanamycin-containing plates for overnight colony growth Day 2 Pick a colony from the agar plate, inoculating it into 5 ml of kanamycin-containing LB broth overnight. This will amplify the plasmid so we can subsequently purify it. Kinetics of acid phosphatase-catalysed conversion of p-NPP substrate (a time course and then measuring the rate of reaction at varying substrate concentrations) Day 3 Purify plasmid from bacteria using a mini-prep kit Quantify plasmid DNA and use as a substrate for benzonase assays Vary conditions of benzonase reactions to determine optimum conditions for the enzyme metal ion concentration, pH, and temperature Run DNA agarose gels of benzonase-digested samples, take images. 3 Background Bacterial transformation (Day 1) Transformation is the process of introducing foreign plasmid DNA into bacteria, with the intention of either expressing a protein for purification or as in this case, propagating the plasmid in bacteria then purifying the plasmid for use in other applications. We will be using DH5 E.coli cells, which are non-pathogenic and which have been made chemically competent that is, simply adding a salt solution (KCM, standing for potassium, calcium, magnesium) allows the bacteria to open up their cell wall pores to let the plasmid in. The plasmid DNA contains an kanamycin-resistance gene (AmpR) and it is on this basis that we select for transformants, adding kanamycin to both the solid agar and liquid growth media. Following transformation, the bacteria are plated on to LB agar with kanamycin, and the following day will form single discreet colonies, all of which are the result of a single bacteria taking up the plasmid. These colonies are picked and inoculated into LB liquid broth containing kanamycin overnight, allowing the bacteria to grow, continually replicating the plasmid and providing us with a reasonable plasmid DNA yield after bacterial lysis and DNA purification. Enzymes An enzyme is a protein with catalytic activity, which speeds up reactions that otherwise would happen very slowly indeed. Each enzyme has an optimal set of conditions under which its activity is maximal this can sometimes be within a very narrow temperature and pH range. Therefore, altering factors including temperature, salt concentration or pH can significantly affect the activity of the enzyme. This can be due to changes to charged active site amino acids important for substrate binding or catalysis, though at more extremes of temperature or pH the enzyme may be denatured and inactivated. Therefore, a steady pH and temperature are important when measuring the rate of an enzymecatalyzed reaction. The rate (V) of an enzymatic reaction is proportional to the substrate concentration up to a certain point, beyond which all the enzyme molecules are busy with substrate, and increasing substrate concentration further does not increase the reaction rate. Under these conditions, the enzyme is termed saturated. Therefore, the rate of an enzymatic reaction is determined during the early phase of the reaction called the initial velocity, or V0. The enzyme rate at which this occurs is called Vmax, for maximum velocity (or maximum rate). The Km is the substrate concentration that under a defined set of conditions gives a rate that is 50% of Vmax. By creating a reciprocal LineweaverBurke plot of the inverse of the reaction rate at a series of increasing substrate concentrations, we can determine both the Km and Vmax values for the enzyme. 4 Acid Phosphatase Experiment (Day 2) Acid phosphatase is an enzyme with phosphomonoesterase activity, catalysing the hydrolysis of phosphate groups from biological macromolecules. In human cells acid phosphatase is present in lysosomes, functioning when these fuse with endosomes. The endocytosis of cell surface macromolecules involves trafficking through endosomes before endosome-lysosome fusion then the contents are degraded. Since endosomes become progressively acidified during function, the enzyme functions best at low pH. A number of compounds have been developed that are converted to a chromophore (a light-absorbing group) upon hydrolysis of the phosphate moiety. These are therefore excellent substrates for measuring the kinetics of acid phosphatase. In our practicals we are using the compound para-nitrophenylphosphate (p-NPP) which itself has no absorbance in the visible spectrum (it absorbs in the UV range). However, one of the hydrolysis products of p-nitrophenylphosphate is para-nitrophenol, which under alkaline conditions is converted to a p-nitrophenolate ion, which is yellow and can therefore be detected using a spectrophotometer. This absorbance provides a means to assess the concentration of reaction product, and hence the rate of reaction (conversion of X product per unit time). Hence, phosphatase activity can be monitored by determining the increase in absorbance at 405 nm that results from the release of the p-nitrophenolate ion. We will get a rough idea of V0 at a fixed substrate concentration (substrate concentration can also be shown as [S]), by carrying out a time course of substrate conversion at a fixed substrate concentration and plotting Absorbance vs Time the gradient of the initial slope of the graph as shown below is roughly equivalent to initial rate of reaction. However, if we want to quantify how an enzyme behaves over a range of substrate concentrations, it is much more accurate to measure the rate of reaction at a range of different substrate concentrations, using the Beer-Lambert Law to calculate the approximate concentration of the converted substrate. This law states that: Absorbance = CL is the extinction coefficient for the compound, C is the concentration and L is the pathlength of the spectrophotometer being used. The extinction coefficient is a parameter that defines how strongly a substance (the product para-nitrophenol) absorbs light at a certain wavelength (405 nm) at a given concentration (per 1 M). Once we have determined the concentration of product obtained at each substrate concentration, we can determine the rate of reaction V by factoring in the reaction time. We will use this data to plot a graph of V against [S] this is called the MichaelisMenten plot. We could use this graph to estimate Km and Vmax, though the method is rather inaccurate. 5 Michaelis-Menten Equation and Graph Instead, rearranging the Michaelis-Menten equation to include reciprocal values allows us to plot a graph of 1/V against 1/[S]. Now instead of a curve, we are working with a linear slope this graph is called a Lineweaver-Burke plot (below). The slope of the line is equal to Km/Vmax, the y-intercept is equal to 1/Vmax and, if we extrapolate the line, the x-intercept is equal to -1/Km. The use of the double reciprocal plot yields much more accurate values for Km and Vmax than an interpretation of the Michaelis-Menten curve. Lineweaver-Burk Equation and Graph 6 We will therefore be performing the following experiments: A) Timecourse of enzyme reaction: measuring the amount of converted product after various amounts of time at a fixed substrate and enzyme concentration B) Calculating the rate of reaction at various increasing concentrations of substrate at a single timepoint and fixed enzyme concentration At the end of each reaction, we will add alkaline NaOH this serves two purposes. Firstly, it increases the pH, producing the alkalinity required to produce the visible colored nitrophenolate ion, which we use to measure concentration. Secondly, it stops the reaction by denaturing the enzyme. The Practical Report will be based in part on these graphs and calculations. Mini-prep (Small-Scale Plasmid Preparation Day 3) We will be purifying plasmid DNA using a spin column-based kit from Qiagen. The columns contain a silica membrane that binds up to 20 g DNA but only in the presence of a high concentration of chaotropic salt (chaotropic agents disrupt hydrogen-bonding). The bacterial cells are resuspended, RNA digested, then the cells are lysed and proteins and lipids precipitated out of solution. This is centrifuged, forming an unwanted white pellet. The clear supernatant contains the DNA, and this is spun onto the column under high-salt conditions. While bound, the DNA is then washed with two wash buffers, then the column is dried by centrifugation. The column also allows simple elution of DNA in a small volume of low-salt buffer at the end of the wash steps. This method eliminates the more traditional time-consuming method of phenolchloroform extraction and alcohol precipitation. Benzonase Experiment (Day 3) Benzonase is a genetically engineered endonuclease from the rod-shaped gram-negative bacteria Serratia marcescens. The protein is a dimer of 30 kDa subunits, which degrades DNA and RNA (regardless of whether it is single stranded, double stranded, linear or circular). It is effective over a wide range of conditions. Benzonase completely digests nucleic acids to small 3-5 bp oligonucleotides. This is ideal for removal of nucleic acids from recombinant proteins and for applications where complete digestion of nucleic acids is desirable. Pre-treatment of a protein sample improves its appearance on a western blot, by eliminating any bound nucleic acids. We will be digesting the plasmid DNA generated during the practical using benzonase, varying metal ion concentration, temperature and pH to determine approximate optima. The DNA will be run on an agarose gel to determine the extent of its degradation this is a crude readout of enzyme activity. An uncut sample of plasmid will be run alongside as a comparison. 7 Day 1 Bacterial Transformation We are using chemically competent E. coli cells to amplify a plasmid that we will later use as a substrate for the Benzonase nuclease. 1. Pre-warm 1 ml LB broth to room temperature 2. You will be provided with a freshly-thawed vial of competent bacteria. This is to be kept on ice. For future reference, competent bacteria must be kept at -80 degrees C and thawed slowly on ice. 3. You have been provided with 2 l pDEST-myc plasmid DNA at a concentration of 200 ng/l. This plasmid contains an kanamycin resistance gene. Make a solution of plasmid DNA at a concentration of 10 ng/l by adding sterile water to the concentrated DNA. How much would you add? 4. Add 2 l (20 ng) of plasmid to the vial of bacteria on ice. Flick once to mix. Wait 5 mins (still on ice) you have been provided with a timer. 5. Add 100 l water, then 10 l KCM mix. Mix by gentle agitation. Incubate on ice for 20 min, then at room temperature for 10 min. 6. Add 700 l of LB broth to the transformed bacteria. Incubate at 37 degrees with shaking for 1 hr. 7. Once the bacteria have grown, we must plate them out so that single colonies will form by tomorrow. The already-melted LB solid agar is being kept warm in a 60- degree water bath. Take a Falcon tube containing 20 ml of agar, and add kanamycin at 50 g/ml. The kanamycin stock provided is at 50 mg/ml how much do we need to add to the agar? Once the kanamycin is added, pour the agar into a Petri dish, label with your name and allow the agar to set fully. 8. Spin the bacteria at 2000 rpm for 1 min. Remove supernatant, leaving approx. 50 l broth in the tube. 9. Resuspend pellet by pipetting up and down in the residual broth. 10. Add 30 l bacteria to the agar plate. 11. Spread the transformed bacteria with the spreader provided, and leave inverted overnight (16 hrs) at 37 degrees C. 8 Day 2 KINETICS OF ACID PHOSPHATASE HYDROLYSIS OF p-NPP Part 1: Obtaining a curve of Product vs Time In this experiment, we are setting up a large reaction with enough volume for removal of multiple samples at different times we will be measuring reaction progress for 30 minutes. Every time we need to take a sample, we will remove some reaction mix and add it to a cuvette containing NaOH to stop the reaction and generate the coloured product. 1. Add 3 phosphatase substrate tablets to ( ) ml of Phosphatase Reaction Buffer (PRB). Each tablet has a mass of 5 mg. The molecular mass of the substrate is 371. Determine how much PRB to add to the tablets to obtain a stock concentration of 10 mM. 2. Prepare a set of 8 cuvettes for the kinetic analysis of purified Acid Phosphatase. Label these cuvettes as follows: No enzyme, 0 min, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min. Add 100 l of 2M NaOH to each cuvette. Set your spectrophotometer to 405 nm. 3. This part of the practical uses a fixed substrate concentration of 1 mM. The reaction will be carried out in PRB buffer, in a final volume of 8 ml, and at each timepoint some reaction mix will be removed and added to the NaOH-containing cuvette on ice to stop the reaction and generate a coloured product, giving us a quantifiable measure of the extent of the reaction. Here we will find out the best timepoint to use in our subsequent experiments we do not want too low or too high an absorbance reading when we vary substrate concentration in the next experiment. Please calculate what you think the composition of the reaction should be, based on the following information and thereby fill in the blanks in Step 4. Once you have done this, please wait. When everyone has finished calculating, we will check the answer and proceed. The enzyme concentration in the final reaction mix should be 0.015 units/ml. The stock enzyme solution provided is 0.15 units/ml. The substrate concentration in the final reaction mix should be 1 mM. The stock concentration you have made up is 10 mM. Each reaction will be carried out in 1 ml volume, and there are 8 reactions (one for each time point plus some extra), so the final volume of the main reaction mix will be 8 ml. The solution should be made up to the final volume with PRB buffer. 4. Set up the solution as indicated, but do NOT add the enzyme to the mix until you are ready to start your timer! 6.4 9 Phosphatase Reaction Buffer ml pNPP (from 10 mM stock) ml WAIT! Dont add the enzyme until you are ready. Enzyme solution ml 4. Set up a No enzyme solution of 900 l PRB and 100 l pNPP and add 900 l of the mixture to a cuvette containing 100 l NaOH. This will act as an initial value. 5. Once you are ready, add the enzyme to start the reaction from Step 4, mix by inversion and start the timer. Work together to make the following process as easy and accurate as possible. 6. At the appropriate time points take 900 l from your reaction mix and add it to the corresponding cuvette containing the NaOH. Place the tubes on ice. This will stop the reaction and create a coloured product. At the end of the 30 min, you can read all your samples sequentially in the spectrophotometer provided. Note the OD405 for each sample in the table provided. Time (min) OD @ 405 nm Transfer the values observed onto the graph paper provided as accurately as possible. The x-axis will be time (min) and the y-axis will be A405nm. Try and draw a curve based on the values. Based on this graph, choose the most appropriate timepoint for the next part of the experiment in which you will vary the substrate concentration and measure the reaction rate. To do this, we could choose the timepoint at which the OD405 was closest to 0.5 as we do not want the absorbance curve to go outside the linear range (a maximal absorbance value of 1). The concentration of substrate in the first experiment was 1 mM based on this timepoint, we can vary the substrate concentration below and above 1 mM to achieve a series of values still in the linear range. Chosen timepoint ( ) min 6.4 0.8 0.8 0 1 25 10 20 30 0.110 0.48 0.70 1.06 1.82 1.63 1.63 maybe 1 as i am not sure 10 Part 2: Determining the Km and Vmax of acid phosphatase 1. Prepare 8 cuvettes, each containing 100 l 2M NaOH 2. Calculate the volume of 10 mM substrate stock solution required to prepare the concentrations indicated in the table below, and the volume of phosphatase buffer required to make the total solution volume up to 1 ml. 3. Make up the following solutions listed in the table below in 8 Eppendorf tubes, labelled appropriately, but do not add the enzyme until you are ready to start the timer, then add the enzyme as quickly as possible to all the tubes and start the timer. The reaction will be allowed to proceed for min at room temperature the time that you chose in the last part of the practical its up to you! Tube Number Substrate Concentration (mM) Vol. substrate Stock (l) Vol. E Stock (l) Vol Phosphatase Buffer (l) 1 0 100 2 0.1 100 3 0.2 100 4 0.4 100 5 0.8 100 6 1 100 7 2 100 8 4 100 4. After the designated time, add 900 l of each solution to the corresponding cuvette containing NaOH, and place the cuvettes on ice. 5. Read Absorbance at 405 nm in the spectrophotometer. Record the results in the Absorbance column of the following table. These will be used to plot a MichaelisMenten graph, then calculate 1/V and 1/[S] to produce a Lineweaver-Burk graph and calculate Km and Vmax. Calculations advice is below. [S] (mM) Abs@405nm (units) [Product] mM Rate V (mM/min) Rate V (m/min) 1/V 1/[S] 0.1 0.2 0.4 0.8 1 2 4 0 10 20 40 80 100 200 400 900 890 880 860 820 800 700 500 0.72 0.48 0.94 1.52 0.97 1.57 1.57 11 Calculations Help 1. Use the Beer-Lambert law (found in the introduction section of this manual) to calculate the concentration of p-nitrophenolate product formed in each tube. The MILLIMOLAR Extinction coefficient of p-nitrophenolate is 18 cm-1. The path length is 1 cm. 2. Use the concentration obtained from Step 1 to calculate the rate of product formed per minute (in mM/min units) 3. Plot substrate concentration [S] against the rate V in Excel or an equivalent program. Use this graph to crudely estimate the values of Vmax and Km. 4. Calculate the values for 1/[S] and 1[V] for each concentration of S. Plot these on a graph in an appropriate program such as MS Excel. 5. Plot the line of best fit for the values obtained. 6. Based on this, the Y-intercept is equal to 1/Vmax. Both X and Y intercepts can be determined using the INTERCEPT function in Excel and highlighting the appropriate graph points. 7. Since Step 5 provides the Vmax value, we can now calculate the Km, as the gradient of the line equals Km/Vmax. The X-intercept also equals -1/Km Colony picking If you have no colonies from yesterday, please pick one from another students plate! 1. Add 5 ml LB broth to a 50 ml Falcon tube. Add 5 l Kanamycin (50 mg/ml stock) 2. Pick a colony from the plates using a pipette tip to inoculate LB broth. Shake overnight at 37 degrees. 12 Day 3 Plasmid Preparation Use the Qiagen MiniPrep Spin kit to prepare a sample of purified DNA for subsequent digestion. A summary of the manufacturers protocol is provided below. Solutions are pre-aliquoted on the bench for each pair. 1. Centrifuge bacteria at 2500 rpm for 10 minutes in the 50 ml tube. Please make sure that the centrifuge is balanced! You should get a nice pellet of bacteria. 2. Pour off the supernatant in one fluid motion into the bleach-containing 50 ml tube provided. 3. Use a 1 ml pipette to aspirate the remaining supernatant and add to the bacterial waste tube. Keep the pellet intact. 4. Add 250 l Buffer P1 to the pellet and resuspend using a 1000 l pipette tip. Ensure that no clumps remain. This solution contains RNase. 5. Transfer 250 l of the bacterial suspension to a clean labeled 1.5 ml Eppendorf tube. 6. Add 250 l Buffer P2. Close lid and invert 10x. 7. Add 350 l Buffer N3. Close lid and invert 10x, or until blue colour disappears completely. A white precipitate will appear (this is unwanted protein/lipid) 8. Wait until everyone has his or her sample ready. Spin the tube at 13,000 rpm in a benchtop centrifuge for 10 min. 9. Remove 700 l supernatant and transfer to the Qiagen Spin Column. Centrifuge at 13,000 rpm for 30 seconds 10. Discard flow-through and replace the spin column into the collection tube. The DNA is now bound to the column. 11. Add 500 l Buffer PB to the column and spin at 13,000 rpm for 30 seconds 12. Discard flow-through and replace the spin column into the collection tube. 13. Add 700 l Buffer PE to the spin column and spin at 13,000 rpm for 30 seconds. 14. Discard flow-through and replace the spin column to the collection tube. 15. Dry the column by spinning at 13,000 rpm for 2 minutes. 16. Remove the spin column, and transfer to a clean, labeled Eppendorf tube. The collection tube with residual PE buffer can be discarded. 17. Add 50 l Elution Buffer to the column directly; making sure that the membrane is soaked. Wait for 2 minutes. 18. Elute DNA by centrifuging at 13,000 rpm for 1 minute. 19. The instructor for the session will quantify the DNA in each prep before continuing with the final experiment. 13 Benzonase Assays Benzonase digests DNA and RNA. We will incubate plasmid DNA with benzonase, varying the conditions to discover whether the enzyme activity is affected by either temperature, metal ion concentration and pH. To avoid running too many gels, each student pairs will be allocated one of these sets of conditions. The idea is to discover how benzonase is affected by altering these variables. Timecourse Variable Calculate the amounts of Tris and MgCl2 required to make 1 ml of 2x BRB (benzonase reaction buffer). This is the solution that will provide a specific set of reaction conditions in the benzonase digestion experiments. Note that this is a 2x stock, meaning that it is double-concentrated and will be diluted 1 in 2 in the final reaction mix. The stocks of Tris and MgCl2 are both 1M. Benzonase Reaction Buffer Volume needed (l) 20 mM Tris-HCl 4 mM MgCl2 Water 1. Label Eppendorfs with the following times, Uncut, 1 min, 5 min, 10 min, 20 min. 2. Calculate the composition of the final reaction mix below. This will contain the provided plasmid DNA, the Benzonase Reaction Buffer, and the Benzonase enzyme. The final volume should be 25 l and the amount of enzyme in each reaction should be 0.01 units 3. Make up the following solution in each tube. Do not add the benzonase until you are ready to start the timer and proceed. Volume (l) 2 g DNA 2xReaction Buffer Water Benzonase (stock is 0.001 units/l) Total 25 l 4. Add the indicated amount of benzonase to each tube, apart from the uncut tube (to this tube add the equivalent volume of water). START YOUR TIMER. 5. At the appropriate time, stop the reaction by adding 10 l 500 mM EDTA to the correct tube and placing on ice. 6. Add 8 l DNA loading dye to each sample. Load 20 l of each on to the DNA gel in consecutive lanes. Dont forget to include the uncut control. 14 7. After the gel has run we will be analyzing it on David Pryces gel imager on Level 3 to do this we will go up in groups. Temperature Calculate the amounts of Tris and MgCl2 required to make 1 ml of 2x BRB (benzonase reaction buffer). Note that this is a 2x stock, meaning that it is doubleconcentrated and will be diluted 1 in 2 in the final reaction mix. The stocks of Tris and MgCl2 are both 1M. Benzonase Reaction Buffer Volume needed (l) 20 mM Tris-HCl 4 mM MgCl2 Water 1. Label Eppendorfs with the following times, Uncut, 4, Room Temp, 37 and 65 degrees. 2. Calculate the composition of the final reaction mix below. This will contain the provided plasmid DNA, the Benzonase Reaction Buffer, and the Benzonase enzyme. The final volume should be 25 l and the amount of enzyme in each reaction should be 0.01 units 3. Make up the following solution in each tube. Do not add the benzonase until you are ready to start the timer and proceed. Volume (l) 5 g DNA (stock is 2 mg/ml) 2xReaction Buffer Water Benzonase (stock is 0.001 units/l) Total 25 l 4. Add the indicated amount of benzonase to each tube, apart from the uncut tube. (to this tube add the equivalent volume of water). Place at the correct temperature either on ice, at room temperature, or in the heat block at 37 and 65 degrees. START YOUR TIMER. 5. After 20 min, stop the reaction by adding 10 l 500 mM EDTA to the tube and placing on ice. 6. Add 9 l DNA loading dye to each sample. Load 20 l of each on to the DNA gel in consecutive lanes. Dont forget to include the uncut control. 7. After the gel has run we will be analyzing it on David Pryces gel imager on Level 3 to do this we will go up in groups. 20 4 976 2.5 12.5 10 15 Presence of metal ions Here we are assessing the role of metal ions in benzonase catalysis. Calculate the amounts of the chemicals required to make 1 ml of several different 2x BRBs (benzonase reaction buffers). Here, each different buffer contains a different metal ion for each reaction. Note that these BRBs are a 2x stock, meaning that it is double-concentrated and will be diluted 1 in 2 in the final reaction mix. The stocks of Tris and MgCl2 are both 1M, the EDTA stock is 500 mM. The NaCl stock is 2M Benzonase Reaction Buffers No Metal Ions Volume needed (l) 20 mM Tris-HCl Water Mg2+ 20 mM Tris-HCl 4 mM MgCl2 Water EDTA only 20 mM Tris-HCl 100 mM EDTA Water NaCl 20 mM Tris-HCl 400 mM NaCl Water 1. Label Eppendorfs with the appropriate labels (Uncut, No ions, EDTA only, MgCl2, and NaCl). 2. Calculate the composition of the final reaction mix below. This will contain the provided plasmid DNA, the Benzonase Reaction Buffer, and the Benzonase enzyme. The final volume should be 25 l and the amount of enzyme in each reaction should be 0.01 units 16 3. Make up the following solution in each tube. Do not add the benzonase until you are ready to start the timer and proceed. Volume (l) 5 g DNA (stock is 2 mg/ml) 2xReaction Buffer Benzonase (stock is 0.001 units/l) Water Total 25 l 4. Add the indicated amount of benzonase to each tube, apart from the uncut tube. To this tube add the equivalent volume of water. 5. After 20 min, stop the reaction by adding 10 l 500 mM EDTA to the tube and placing on ice. 6. Add 9 l DNA loading dye to each sample. Load 20 l of each on to the DNA gel in consecutive lanes. Dont forget to include the uncut control. 7. After the gel has run we will be analyzing it on David Pryces gel imager on Level 3 to do this we will go up in groups. Variable pH Here we are assessing the role of pH in benzonase catalysis. Calculate the amounts of the chemicals required to make 1 ml of several different 2x BRBs (benzonase reaction buffers). Here, each different buffer will impart a different pH to the reaction. Note that these BRBs are a 2x stock, meaning that it is double-concentrated and will be diluted 1 in 2 in the final reaction mix. The stocks of Tris and MgCl2 are both 1M, the stocks of citrate and borate buffers are 0.1M each. Benzonase Reaction Buffers Volume needed (l) Low pH Buffer (Citrate, pH 4.3) 20 mM Citrate Buffer 4 mM MgCl2 Water Neutral pH Buffer (Tris, pH 7.4) 20 mM Tris-HCl 4 mM MgCl2 Water 17 High pH Buffer (Borate pH 9.8) 20 mM Borate Buffer 4 mM MgCl2 Water 1. Label Eppendorfs with the appropriate labels (Uncut, pH4, pH7, pH10). 2. Calculate the composition of the final reaction mix below. This will contain the provided plasmid DNA, the Benzonase Reaction Buffer, and the Benzonase enzyme. The final volume should be 25 l and the amount of enzyme in each reaction should be 0.01 units 3. Make up the following solution in each tube. Do not add the benzonase until you are ready to start the timer and proceed. Volume (l) 5 g DNA (stock is 2 mg/ml) 2xReaction Buffer Water Benzonase (stock is 0.001 units/l) Total 25 l 4. Add the indicated amount of benzonase to each tube, apart from the uncut tube. Add the equivalent amount of water to this uncut tube. 5. After 20 min, stop the reaction by adding 10 l 500 mM EDTA to the tube and placing on ice. 6. Add 9 l DNA loading dye to each sample. Load 20 l of each on to the DNA gel in consecutive lanes. Dont forget to include the uncut control. 7. After the gel has run we will be analyzing it on David Pryces gel imager on Level 3 to do this we will go up in groups. The End!

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