Documents about Phosphate Buffer
Lab 6
Cal Poly Pomona, MIC 428
Excerpt: ... ill be used in enzyme experiments. -To determine an enzyme (glucose-6-P-dehydrogenase) as an example of how to study a metabolic pathway in bacteria. EXPERIMENTAL PROCEDURE Part I (day 1): Cell-free extract preparation for enzymatic studies. Mechanical or chemical methods have to be employed in order to release intracellular enzymes. We will use a physical method to break the cells. Procedure (experiment is performed per group of 4-5 students): 1. Place 40 mL of E. coli culture in each of 2 centrifuge tubes. 2. Balance the tubes and recover the cells by centrifugation in the cold at 10,000 g/5 min. 3. Remove supernatant and resuspend the pellet in 10 mL of phosphate buffer 4. Pool the suspended pellets in one centrifuge tube and add an additional 20 mL of phosphate buffer . Centrifuge the suspended pellets in the same conditions as in 2. 5. After centrifugation, discard the supernatant and weigh the centrifuge tube containing the cell pellet. Suspend the pellet in 5 mL of phosphate buffer and transfer the susp ...
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PPOInApples
East Los Angeles College, U 0000504
Excerpt: ... Polyphenoloxidase Activity In Apples Aim a) To prepare an enzyme extract from apples. b) To measure the polyphenoloxidase activity of the extract. c). To investigate the effect of various inhibitors. Materials Apples of various varieties Acetone Substrate, Catechol, 0.0lmol l-1 +proline, 0.0lmol l-1 in phosphate buffer , 0.1 mol l-1, pH = 7.0 phosphate buffer , 0.1 mol l-1, pH = 7.0 Inhibitors Citric acid (0.02 M) Ascorbic acid (0.01 M) Sodium metabisulphite (0.01 M) Cysteine (0.01 M) EDTA (0.01 M) Sodium chloride (0.1 M) Cinnamic acid (0.01 M) p-Coumaric acid (0.01 M) Ferulic acid (0.01M) Hydrocinnamic acid (0.01M) Method a) Extraction Homogenise l0g of apple with 50 ml of acetone for 1 minute. Filter the homogenate under suction through a sintered glass filter, porosity 4. Wash the residue with 10 x 10 ml aliquots of acetone. Carefully transfer all the homogenate to a 100 ml beaker and place in a vacuum desiccator. Place under vacuum for 20 minutes. This sample should be virtually free of ...
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CHM 343 Lab Report 3
Cal Poly Pomona, CHM 343
Excerpt: ... ent ratio was set up as 40% phosphate buffer and 60% methanol. The sample was injected and run. All the three samples of ascorbic, benzoic acid and caffeine were run separately, followed by the unknown sample. The data collected from the peaks were analyzed for their concentrations. Results: (To figure out the amount of each component in the unknown in ppm, we must first know the ppm of each of the standards.) Table 1.1: Results of each standard run Name: Ascorbic Acid Caffeine Benzoic Acid Unknown # 1 Unknown # 2 Retention Time: (minutes) 2.558 3.350 4.646 3.370 4.938 Area: (mAU) 3406.48486 1280.95178 225.42961 139.10123 13.557650 Width: (mAU) 4.97104e4 1.94081e4 4266.70898 2111.27417 396.26660 Seeing as how the retention times of both of the unknown peaks were very similar to caffeine and benzoic acid, respectively, the ppm of only those two are needed. No further calculations on Ascorbic Acid are needed. 4 CHM343 Lab Report # 3 Choon Liang Tan Ppm of Standard Solutions: Table 1.2: Prop ...
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ChapterThreeFigures26to28
Virginia Tech, LIB 12012000
Excerpt: ... Figure 26. Chemical stability studies: Chemical treatment and effect on pressureflow. A 90 1.6 cm column bed of 3.5 wt. % CL-DEAE cellulose support was flow-packed with 3 CV of 15 mM sodium phosphate buffer (NAP), pH 7.8 at 10 cm/min and conditioned with 0.5 N NaOH (1 CV) at 5 cm/min. Column Backpressures were measured at the end of steps a, c and d in the cleaning cycle. Cleaning cycles: (a) 4 M NaCl (2 CV) at 10 cm/min; (b) 15 mM NAP (2 CV) at 10 cm/min; (c) 0.5 N NaOH (1 CV) at 5 cm/min; and (d) 15 mM NAP (2 CV) at 10 cm/min. 104 Figure 27. 3.5 wt. % beaded cellulose support. Magnification: 50. The uniform large interstitial spaces allow for low column backpressures at high linear velocities (~ 0.05 PSI/cm at 10 cm/min, see pressure stability studies section). Note the low surface area to volume ratio. 105 Figure 28. DEAE FF-Sepharose support. The wide range in bead particle diameter sizes and small interstitial spaces result in relatively high column backpressures (~ 0.9 PSI/cm at 10 cm/min, s ...
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Status_05.16.2006
Oregon State, CHE 415
Excerpt: ... Weekly Status Report Citrate It http:/classes.engr.oregonstate.edu/che/spring2006/che415/citrateit/index.html Submitted May 16, 2006 Author: Jeremy Campbell Team Members: Jeremy Campbell, Travis Bolsinger, Eric Andersen Previous Week Accomplishments Crosslinking reactions were modified to include a large excess of EDC and buffered to an acidic pH of 4.8. Visible changes took place as the EDC was converted to product urea. Visible changes place the reaction time on the order of minutes. Changes were made to reduce variability in test results and eliminate possible influences of error. Non reacted carboxylated beads were washed with phosphate buffer to mimic the processing steps that reacted beads are subjected to and to align tests with the phosphate buffer control times. Plasma in active testing was placed in an ice bath to provide temperature control and eliminate drift in clotting times observed over the course of sampling. Polyacrylate/ phosphate buffer solutions were tested to investigate the observed act ...
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exams
Georgia Tech, CHEM 4582
Excerpt: ... protect scientists form exposure to x-rays during an xray diffraction experiment? More than one answer may be correct. a. Wearing safety glasses b. Keeping sufficient distance from the x-ray beam c. Use of lead-containing shields d. Wearing a lab coat e. Wearing a radiation badge Heme is an achiral molecule that binds to the protein myoglobin. However, a 30 M solution of myoglobin (containing heme) in 5 mM sodium phosphate buffer at pH 7 exhibits a positive circular dichroism signal near 400 nm (where heme absorbs). Since chirality is a requirement for circular dichroism, what is responsible for the CD signal? Which of the following components is required for a fluorometer? a. monochromator b. Xe lamp c. Circular cell d. Goniometer e. Detector 5. 6. 7. 8. 9. Why is it important to purge a CD spectropolarimeter with nitrogen prior to measuring CD spectra? The absorption of a macromolecule with a molar extinction coefficient of 1000 M-1 cm-1 is measured in a 1cm path length cell. The OD280 is 0.5. What is ...
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651_EZA_Prelab_Lecture_2009
Wisconsin, BIOCHEM 651
Excerpt: ... effects Stabilizing agents (detergent, salt, reducing agent, etc) d i t t ) Factors affecting an enzyme assay: Factors affecting an enzyme assay: The Assay Construct a standard curve for gal Study the kinetics of gal Study the kinetics of gal The Assay Colorimeter: Beer's law: A=bc A = absorbance (400 nm) = extinction coefficient b = path length (typically 1 cm) c = concentration The Assay Colorimeter Sensitivity: Colorimeter Sensitivity: 2 A Absorbance e Units 1.5 1 0.5 0 Ideal Real Question 4 Working Range W ki R The Assay Materials: gal: gal is a powder. (2.6 mg protein/3 mg solids) (Stored at 20 C) l d ( / ld )( d ) Stock solution is prepared by dissolving gal in 1X phosphate buffer + 25% glycerol. (3 mg solids/mL) (Flash frozen in Liq. N2 and stored at 80 C) On experiment day, the stock is diluted 200X in 1X phosphate buffer . (15 g solids/mL) (Keep on ice) 1X Phosphate buffer (50 mM sodium phosphate buffer , pH 7.0) PNPG (0.32 mM in 1X phosphate buffer ) Sodium carbonate ...
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ListofTables
Virginia Tech, LIB 12012000
Excerpt: ... List of Tables Chapter Three Table 1. Pulse-flow transport studies with 2 wt. % cellulose supports Table 2. Pulse-flow transport studies with 2 wt. % CL cellulose supports Table 3. Pulse-flow transport studies with 2 wt. % CL-DEAE cellulose supports Table 4. Pulse-flow transport studies with 6 wt. % cellulose supports Table 5. Pulse-flow transport studies with 6 wt. % CL cellulose supports Table 6. Pulse-flow transport studies with 6 wt. % CL-DEAE cellulose supports Table 7. Pulse-flow transport studies with 10 wt. % cellulose supports Table 8. Pulse-flow transport studies with 10 wt. % CL cellulose supports Table 9. Pulse-flow transport studies with 2 wt. % CL-DEAE cellulose supports Table 10. Dynamic BSA binding capacities of 2 wt. % CL-DEAE cellulose supports using 50 mM tris buffer, pH 8.6 with varying concentrations of NaCl Table 11. Dynamic BSA binding capacities of 2 wt. % CL-DEAE cellulose supports using 15 mM sodium phosphate buffer , pH 7.8 with varying concentrations of NaCl Table 12. Dynamic BSA b ...
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Exp114
Penn State, CHEM 35
Excerpt: ... potassium permanganate (KMnO4) in a small beaker with water, you would notice the purple color of the potassium permanganate. If you then added a reducing agent, such as sodium dithionite (sodium hydrosulfite, Na2S2O4), dropwise to the beaker, you would observe a color change from purple to pale pink as the permanganate ion is reduced to Mn2+ and the dithionite is oxidized to sulfite SO32-. Write and balance the redox equation for this chemical reaction. Calculate the weight of potassium phosphate monobasic (KH2PO4; FW = 136) and potassium phosphate, dibasic (K2HPO4; FW = 174) required to prepare 20 mL of a pH 6.8 phosphate buffer solution that is 20 mM in potassium phosphate monobasic (KH2PO4) and 20mM in potassium phosphate, dibasic (K2HPO4). Cautions The isolation of myogolbin requires the use of a bench-top centrifuge. Be sure to balance the centrifuge with two tubes of equal weight when operating, or damage can occur to the centrifuge and the centrifuge tube may shatter. Potassium ferricyanide is a har ...
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Lab07
Creighton, BIO 450
Excerpt: ... your muscle samples. 5. Centrifuge the samples at a speed of 30 for 3 minutes. Important note be sure the centrifuge is balanced by placing paired tubes opposite one another! The substances you will assay are in the resulting supernatant. 6. Draw off about 1.5 ml of each supernatant and put them in separate tubes. Use these supernatant samples for all subsequent work. 7. The full strength supernatant will be used for most of the assays, but you will also need dilute samples for the LDH assay. In new test tubes, create a 5fold dilution of each supernatant sample by adding 200 l of supernatant to 0.8 ml phosphate buffer . This is the sample you will use for the LDH assay. Place all samples on ice. Determining total protein concentrations We will be using a "Total Protein Reagent" to determine the total protein concentrations of our supernatants. This reagent is alkaline and contains copper ions. The reagent reacts with peptide bonds to produce copperprotein complexes, which have a purple color ...
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641prob1
Delaware, CHEM 641
Excerpt: ... e label the useful buffering region of this solution. In terms of equivalents of base added what is the useful buffering region? 5) Suppose you are working in a Biochemistry lab and the pH meter is broken. You urgently need to make a phosphate buffer at pH 7.8. From an inspection of your chemical inventory you note that you have the following forms of phosphate to make your buffer: phosphoric acid (H3PO4), mono-basic sodium phosphate (NaH2PO4), di-basic sodium phosphate (Na2HPO4) and tri-basic sodium phosphate (Na3PO4). You then find in your Biochemistry textbook that the 3 pKas for phorphoric acid are pKa1 = 2.15, pKa2 = 7.2 and pKa3 = 12.4 (a) In order to make 1 L of a 0.1 M sodium phosphate buffer at pH 7.8 how many moles of which compounds would you mix together? Show all your work. (b) If you needed to make a buffer at pH 9.0, would a phosphate buffer be a good choice? Sketch a titration curve of phosphoric acid to help explain your answer. 1 ...
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Expt2
St. Edwards, CHEM 45
Excerpt: ... Chem 4245 Materials: DNA : Purity, properties and ligand binding 1. 1 L of Phosphate buffer (0.01mol L-1 / pH=7.4) prepared as follows : (i) prepare 1L of 0.02 M NaOH(aq) (ii) prepare 1L of 0.02 M sodium dihydrogen phosphate(aq) or (i) prepare 1L of 0.02 M KOH(aq) (ii) prepare 1L of 0.02 M potassium dihydrogen phosphate(aq) (iii) add x mls of (i) to 50 mls of (ii) and dilute to 100 mls to get desired pH: x mls 3.5 pH 5.8 5.8 6.0 9.1 6.2 13 6.4 18 6.6 24 6.8 30 7.0 35 7.2 40 7.4 43 7.6 45 7.8 47 8.0 2. 500 mLs of saline- phosphate buffer made from equal parts: 0.15M NaCl(aq) and 0.01M / pH=7.4 Phosphate(aq) Note : Do NOT use disposable cuvettes Part A.1 Determination of the concentration of DNA sample Procedure: Transfer DNA sample to 10 mls of saline- phosphate buffer and dilute to 25 mls. Using pure saline- phosphate buffer as a reference measure the absorbance in a 1 cm path length quartz cuvette at 260 nm (A260 ) of this solution. If the reading is above 2 dilute sample to obtain reading aroun ...
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DefinedMedium_Rhodo_spp20I24_20KY1
Michigan, MICRO 406
Excerpt: ... DEFINED MEDIUM FOR RHODOCOCCUS SPP. I24 AND KY1 (MEDIUM RARE) (NH4)2SO4 MgSO4.7H2O CaCl2.2H2O MOPS* A9 trace elements solution Stock Solution A 1.0 M phosphate buffer Glucose 1.4 g/l 1.0 g/l 0.015 g/l 1g/l 1.0 ml/l 1.0 ml/l 35.2 ml/l 40 g/l NaMoO4.2H2O 2.0 g FeNa.EDTA 5.0 g filter sterilize; store at 4C FeSO4.7H2O 0.5 g ZnSO4.7H2O 0.4 g MnSO4.H2O 0.02 g H3BO3 0.015 g NiCl2.6H2O 0.01 g EDTA 0.25 g CoCl2.6H2O 0.05 g CuCl2.2H2O 0.005 g filter sterilize; store at 4C K2HPO4 Kh2PO4 113 g 47 g Stock A (per liter of water): A9 trace elements solution: (per liter of water) 1.0 M phosphate buffer : (per liter of water) Note: Add (NH4)2SO4 , MgSO4.7H2O, CaCl2.2H2O and MOPS buffer to 863 ml of water and autoclave. Then add sterile stock A, A9, 1M phosphate and 100 ml of 400g/l glucose solution. *MOPS is optional P. LESSARD 2002, ALL RIGHTS RESERVED ...
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BISC 220 Lab 8 expected results
USC, BISC 220
Excerpt: ... Chapter 8: ELISA technique Specifications Coating Buffer: 0.1% Sodium Bicarbonate, pH 8.6. Coated antigen: Salmonella typhimurium Washing buffer: 0.01M Phosphate Buffer ed Saline pH 7.4 with Tween 20 (.05%) Blocking buffer: 0.01M Phosphate Buffer ed Saline pH 7.4 with 1% Bovine serum albumin (BSA) Primary antibody: Abcam ab35156 rabbit polyclonal IgG (100x dilution in blocking buffer) Enzyme-labeled secondary antibody: Sigma A3687 polyclonal goat anti-rabbit IgG, alkaline phosphatase (AP) conjugated (1000x dilution in blocking buffer) Substrate: BCIP/NBT (alkaline phosphatase; chromogenic color change) BCIP: 5-bromo-4-chloro-3-indolyl phosphate NBT: p-nitroblue tetrazolium chloride. Results Just as the lab manual describes, well 12 (your control) should be regarded as colorless (though color can evolve slowly) because there was absolutely no patient serum added. Since the "patient serum" contains antibody, well 1-11 would show blue color gradually changing from dark to light, to colorless. Note These are fro ...
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Exam_2001
University of Iowa, C 004211
Excerpt: ... 4:211 Midterm exam Name:_ Use only the space provided in the question! Note: you can get 104 pts but it's hard to exceed 95 pts. 11/1/01 Good Luck! 1) The hydrolysis of acetylimidazole was measured at pH 7.0 and pH 7.3 twice: (a) in phosphate buffer and (b) in imidazol buffer. O O N N + H2O _ + HN + NH O (a) k pH 7.3 pH 7.0 k (b) pH 7.3 pH 7.0 [buffer] [buffer] a. (12 pts) Write a reaction mechanism for the reaction in phosphate buffer (a): b. (12 pts) Write a reaction mechanism for the reaction in imidazol buffer (b): -1- 2) (24 pts) An enzyme is catalyzing the following reaction: H 2N N R - O 2C N N + CO2 H 2N N R In its active site the enzyme has lysine, arginine, and tyrosine. Mutation of the tyrosine to phelylalanine result in an inactive enzyme. Mutation of lysine to histidine decreased kcat by a small factor. Mutation of the arginine to lysine result in an inactive enzyme. Suggest a mechanism for the enzymatic reaction: -2- 3) a. (20 pts) Develop the initi ...
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BMI_Lab4Photometry
St. Johns, PHS 2301
Excerpt: ... Laboratory IV Title: Photometry and Writing a Lab Report This lab has two activities. Students must have a laptop for this lab. 2 students per group for Photometry, 1 student per group for Writing a Lab Report. ACTIVITY 1 Topic: Photometry Principle: 0.2M Acetate buffer, pH 4.0 [unionized, weak acid] 0.2M KH2PO4/NaOH buffer, pH 7.4 [partially ionized, weak acid + conjugate base] 0.2M Carbonate buffer, pH 10.0 [ionized, conjugate base] 0.2mM p-Nitrophenol Objective: a) To determine the extinction coefficient or molar absorptivity of p-Nitrophenol. b) To determine the pKa for the dissociation of p-Nitrophenol. Materials: 1. Acetate buffer pH 4 2. Carbonate buffer pH 10 3. Phosphate buffer pH 7.4 4. Distilled water 5. p-Nitrophenol.0.2mM 3 Test tubes 1 Test-tube rack 1 x 1ml pipette 3 x 5 ml pipette 6 Cuvettes (visible range) Marker Pipettor (green) Waste beakers Laptop (students must bring their own) Spectrophotometer Procedure: 1. Check that you have all the material above (except Spectrophotometer which w ...
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lecture 2 outline
UCSC, BIO 100
Excerpt: ... B. Physiological Buffers 1. Intracellular fluids (pH 6.9-7.4) a) phosphate buffer system b) Other ways intracellular pH is buffered 2. Extracellular fluids (pH near 7.4): the bicarbonate buffer system (Box 2-4) ...
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BMI_Lab3PH Buffers
St. Johns, PHS 2301
Excerpt: ... Laboratory # III Topic: pH, Buffers, and Titration Principle: A buffer is a solution that resists pH changes when strong acids or bases are added to the solution. pH is the negative log of H+ ion concentration. Bronsted-Lowry acid-base theory describes acid as a proton (H+) donor and a base as a proton (H+) acceptor) Most buffer solutions consist of a weak acid and its conjugate base (salt of the weak acid). The buffer equation pKa - pH = log weak acid/salt is useful to calculate ratio of acid/salt needed to give required pH . pKa is the negative logarithm of the acid dissociation constant, Ka. Buffers are effective in resisting a change in pH within a range of about 1pH unit above and below their pKa. Buffers of different pH are prepared by mixing stock solutions of the weak acid and a salt of the acid (refer lab2 activity1). The buffer works best when the pH is close to the pKa of the weak acid. pH of blood is maintained by a combination of phosphate and carbonate buffering system Phosphate buffer pH range ...
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photosynthesislabreport
N.C. State, BIO 183
Excerpt: ... Doorhy 1 Colleen Doorhy BIO 183L Sec 219 2-28-07 Comparison of Photosynthetic Rates between Boiled and Unboiled Thylakoids at Varying Light Wavelengths Abstract To determine the photosynthetic rates of a blank and five different treatments including variations of thylakoids and colors of light a photosynthesis experiment was performed. The purpose of this experiment was to verify if boiled or unboiled thylakoids would yield photosynthetic rates and which color of light would also yield the highest photosynthetic rates. A blank test tube with 1.0 mL of phosphate buffer , 3.0 mL of dH 2O, and 1.0 mL of 0.1% DPIP receives white light. Five different treatments were given in separate test tubes to determine the variations of photosynthetic rates at contrasting conditions. The amount of absorbance of each test tube was measure by a spectrophotometer in nanometers (nm). Absorption decreased in each treatment as the time increased at 30 second intervals of being exposed to varying colors of lights. The white li ...
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exp_1_long
Arizona, BIOC 463
Excerpt: ... temperature dependent behavior is most critical for protein or enzyme purification schemes in which both the pH and temperature of the procedure are critical to the isolation of an active form of the protein. There are easy and practical ways of dealing with this problem that will be discussed in class. Finally, these experiments will require the dilution of stock buffer solutions to give a range of concentrations. This will require use of volumetric methods (i.e. pipetting). In most biochemistry laboratories, concentrated solutions of a variety of reagents are first prepared. The researcher then makes the appropriate dilution of the stock solution to give a final working concentration. Therefore, it is extremely important to be able to accurately and reproducibly use a variety of volumetric devices such as graduated cylinders, serological pipettes, and adjustable volume pipettors to make these dilutions. In this set of experiments, we will: 1 Prepare phosphate buffer s with different pH values. ...
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EXAMIIIANswerWEb
UCLA, CHEM 154
Excerpt: ... d in concentrating the protein so that we can test lacR binding at high enough concentrations. The final ammonium sulfate step also aids in removing the concentrated phosphate buffer used to elute the protein. This concentrated phosphate buffer will interfere with lacR binding to the radiolabeled oligo. EXAM 3: Project III 154 Winter 2004 Name:_ TA:_Section:_ 4. (40 pts.) Suppose you set out to test the binding orientation of the recognition helices of the protein, CAP, to DNA using beta-galactosidase assays and suppressor mutant pairs. CAP is dimeric protein with each monomer containing a helix-turn-helix motif (a very similar structure of to a dimer of the lac repressor. In order for CAP to bind the DNA, cyclic AMP must present to induce a conformational change in the structure of CAP. The sequence of the recognition helix is: Arg1-Glu2-Thr3-Val4-Gly5-Arg6-Iso7-Leu8-Lys9 The endogeneous CAP binding site on the DNA has a sequence: 5'-ATGTGAGTTAGCTCACTC-3'. a) Before any ex ...
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Supplement
St. Edwards, CHEM 45
Excerpt: ... o allows for an estimate of the occupancy of the minor groove by calculating the number of nucleotides per ligand bound. NH2 + 2HN + NH2 NH DAPI NH2 Experimental Preparation of Buffer Phosphate buffer (0.01mol L-1 / pH=7.4) prepared as follows : (i) prepare 1L of 0.02 M NaOH(aq) 1 (ii) prepare 1L of 0.02 M sodium dihydrogen phosphate(aq) (iii) add 40 mL of (i) to 50 mL of (ii) and dilute to 100 mL to obtain the desired pH of 7.4. Preparation of Reagents This experiment requires the following solutions : (i) Prepare a solution of 50g/mL of DNA in phosphate buffer by dissolving a 1 mg vial of Calf-Thymus DNA (Sigma-Aldrich) in 20 mL of 0.01 M phosphate buffer of pH 7.4. Confirm the concentration by measuring the absorbance in a 1 cm path length quartz cuvette at 260 nm (A260 ) of this solution. A 50g/mL solution of native double-stranded DNA in a 1cm cell gives a A260 reading of 1 AU . The purity of the DNA can be determined by measuring the absorbance at 280 nm (A280 ). A pure solution of native double ...
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