Introductory Cell Biology Laboratory
Introductory Cell Biology Laboratory LIFE 212
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This 34 page Class Notes was uploaded by Christop Jacobs on Monday September 21, 2015. The Class Notes belongs to LIFE 212 at Colorado State University taught by Safadi-Chamberlain in Fall. Since its upload, it has received 101 views. For similar materials see /class/210120/life-212-colorado-state-university in Life Science at Colorado State University.
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Date Created: 09/21/15
12 September Spectrophotometry Is the most often used theory and technique in a biochemistry lab Is a technique that measures the interaction of electromagnetic radiation with molecules atoms or ions Absorption Emission Fluorence Principle of spectrophotometry Each substance absorbs a unique wavelength of light monochromatic light from the light spectrum Substances can be analyzed qualitatively and quantitatively Substances appear a certain color because they absorb certain wavelengths of light and transmit others The Absorption Spectrum Absorption over a range of wavelength Lmax wavelength at which absorbance is the greatest Lmax is characteristic of each chemical substance and provides information on the electronic structure of the analyte Action Spectrum Is the rate of a physiological activity plotted against wavelength of light BeersLambert Law Direct linear relationship of Absorbance A to concentration C of a solute AELC A absorbance E extinction coefficient C concentration The equation follows the equation of a straight line Y mx b Where m is the slope of the line and b is the y intercept A C E A 55 Note Absorbance has no units Standard Curve Determining the concentration C ofa substance in solution when E is unknown or when a substance does not obey Beer s law Enzyme Kinetics Enzymes run all cell functions Enzyme activity is studied by enzyme kinetics Important in disease diagnosis and research Enzyme Kinetics study of Rate or velocity of an enzyme catalyzed reaction Factors which may affect it Enzymes act as catalysts Enzyme brings the substrates into the proper alignment or configuration to speed up their reaction or conversion into products Enzymes are highly selective in binding of substrates Because of their 3dimensional structure KeyLock mode of enzyme action Steric specificity Binding of substrate to enzyme is in a geometric arrangement Active site of an enzyme The molecular place where the substrate binds and at which catalysis takes place Catalytic groups protein residues that directly participate in the enzymatic reaction Making and breaking of bonds Substrates are bound to enzymes by multiple weak interactions Km Michaelis constant Units of Km are concentrations M Characteristic for an enzymesubstrate interaction under defined pH and temp Gives an idea about the affinity of an enzyme to its substrate important measure of the enzyme rate of reaction Km low high affinity of the enzyme to the substrate Km high low affinity of the enzyme to the substrate Enzymes often have multiple substrates a substrate with low Km will bind more quickly to the enzyme than those with high Km Environment affects enzyme activity Enzymesubstrate bestfit configuration reaction maximum rate Under optimal environmental conditions Factors affecting rate of enzyme action pH Temperature Salts Cofactors and coenzymes Concentration of the substrate Concentration of enzymes pH How does it affect enzyme activity Forces that keep the proteins in a stable conformation and give them their tertiary structure Affects the state of ionization of acidic or basic amino acidsd which affects the 3D conformation of the enzyme Temperature More energetic collisions The number of collisions per unit time will increase The heat of the molecules in the system will increase enzyme denatures Temperature optimum of the enzyme A temperature range in which a maximal rate of reaction is achieved Salts and ions Salting out Change the solubility of the enzyme ons can bind to the enzyme and either enhance or impede substrate binding eg Calcium calmudulin Can be cofactors Cu FeH Can be part of the active site eg iron in cytochrome oxide C copper in Tyrosinase Other factors Coenzymes are organic molecules that are required by certain enzymes to carry out catalysis Bind to the active site but are not substrates eg NAD NADP FAD Coenzyme A coenzyme Q Vit 32 36 312 and Biotin Cofactors inorganic substances that are required for or increase the rate of catalysis Eg Cu Fe Zn Measuring rates of enzyme catalyzed reactions Rate of a disappearance of a particular substrate or rate of appearance of a particular product Experiments Optimum Enzyme Concentration Effect of pH Effect of Temp Objective Find the concentration that produces 2 umoles of product in 3 5 minutes Step 1 Turn on the Specs set the WL to 475 nm blank Step 2 From your graph find the absorbance that corresponds to 2 moles M L calculatemM 1 m concentration Step 3 dilute enzyme concentrations Step 4 set up reaction Step 5 record time to reach the deduced absorbance Step 6 Calculate reaction velocity Effect of pH on rate of reaction Step 1 set up reaction Step 2 add 05 mL enzyme Step 3 Invert time transfer to a cuvette and spec Step 4 Record abs after 15 minutes Step 5 Find corresponding concentration from graph and convert it to mmoles find velocity Plot a curve to find optimum pH for the enzyme Note Start one pH at a time wash cuvette in between Effect of Temp on Rate of Reaction Step 1 Set up reaction 25 mL 8 mM DOPA Tips Step 2 Equilibrate for 5 min before adding the enzyme Step 3 Add the enzyme to one temp at a time incubate for 15 min Step 4 Immediately transfer to the cuvette Step 5 Spec record absorbance Step 6 Use standard curve to find concentration and the umoles Step 7 Plot your data 3 sets of tubes are provided 4 concentration 8 pH 8 temp uM mM M concentration umol mmol mol amounts Blank 25 mL of citrate buffer pH 48 05 mL ofenzyme Plan your time be organized and fast with your partner Temp experiments are time sensitive Make your needed calculations before the experiment Use wash bottle to wash cuvette in between DOPA will spontaneously turn light brown at high pH and temps Keep your parafilm pieces handy for fast inversion of your cuvette Correction Page 56 in manual add 0 C and 44 C 29 August Lab 2 PreLab WriteUp Theory or background information of the use of ELISA EnzymeLinked Immunosorbent Assays for detection of protein antigens Scientific question to be answered Hypothesis and predicted resuIts Objective of this experiment Antibody Antigen protein Interactions A special type of Protein Protein Interaction Antibodies are a type of immuneresponse protein made in vertebrates Used to detect disease pathogens Is used in many applications in research labs Antibodies are proteins that are produced as part of the adaptive immune response to potential pathogens foreign antigens Characteristics of Antibodies Abs Abs are produced on the surface of white blood cells B cells Many types of Ab molecules are produced at different times in the immune response Part of the immunogIobin protein cIass Can be Monoclonal PolycIonaI Antigens are defined as any foreign substance detected by the immune system Each Ab binds to a single Ag binding site epitope Response to a single antigen results in many types of antibodies polycIonaI antibodies Many Assays Use Monoclonal Abs Monoclonal Abs are produced from hybridoma cells They recognize a single antigenic epitope Lab 2 Use an immunoassay to detect the antigenantibody response Immunoassays are highly SPECIFIC and SENSITIVE Specificity is based on the highly precise interaction between antibodies and their antigens AbAg Sensitivity is based on the ability to detect a very few molecules in a complex mixture Therefore high sensitivity is based on a low detection limit Substrate aminosalicyclic acid Also uses enzyme to show a color change Immunoassays use blocking agents Blocking agents Are proteinaceous Bind to nonspecific binding sites Improve binding specificity of Abs Examples normal serum nonfat dry milk casein milk protein bovine serum albumin ELISA Procedure detection of HIV Antibody in Human Blood Immobilize Ag Add mock human serum Which may or may not contain antibodies to HIV Add 2 Ab Linked conjugated to an enzyme Add chromogenic substrate Only serum samples with HIV Ab will react Standards and Controls Controls Establish the range of expected results Positive Indicative of successful enzymeactivated color change Negative Should produce no color change Add confidence that washes were thorough Special considerations Wear gloves Use a waste beaker to collect the liquid at each step Label the small transfer pipettes Use a clean fresh pipette for each new reagent Pipette carefully to avoid cross contamination Wash well but not forcefully To remove extra reagents Fill well completely during wash steps Understand the purpose of controls False PositiveFalse Negatives Misdiagnoses have serious implications for patient care Avoid false positivesfalse negatives Use proper controls Carefully following standardized protocols Avoiding protocols with suboptimal specificity Direct OneStep Detection Disadvantages Lower specificity Higher chance for false positive 7 September Notes Spectrophotometry Employs Beer s Law relates quot39 solution Enzyme kinetics Extraction of enzyme Tyrosinase using high salt precipitation Principles of Spectrophotometry IL T Transmittance U A log10 T A absorbance optical density Monochromatic only absorbing one wavelength Beer Lambert Law Factors affecting Absorbance of solutions Concentration c Pathlength L centimeters Extinction coefficient E also called Molar absorptivity Absorbance of a unit concentration of a solute in a solution depends on the chemical nature of the substance has the units of reciprocal concentration and length M 1 cm l Direct linear relationship of Absorbance to concentration of a solute AELC Ci EL Ei CL Note Absorbance has no units The equation follows the equation of a straight line y mx b M is the slope of the line b is the y intercept Application of Beer s Law Measuring concentrations of chemical substances in solution Determining the extinction coefficient of a substance Standard Curve Determining the concentration ofa substance in a solution where E is unknown or when a substance does not obey Beer s Law Determining the extinction coefficient of a substance by determining the slope Enzyme Kinetics Enzymes catalyze chemical reactions They speed up the rate of a chemical reaction without being used up Each enzyme has specific substrate targets Targets are limited by the molecular structure of the enzyme s active site Enzyme Kinetics The study of the rate of enzymecatalyzed chemical reactions Reagent Blank Contains all ingredients of a reaction except the ingredient that is measured 17 October Principles of Light Microscopy White light source is focused by 3 sets of lenses condenser objective and ocular Magnification is distinct from resolving power resolution Light microscopy magnifies up to 500X over that detectable by human vision Optical Magnification the ratio of the apparent size of an object to its true size Magnification is limited by Lens resolution the ability to resolve detail ie 2 points that are a given distance apart Resolution is limited by diffraction Diffraction is influenced by lens quality NA The Kohler Illumination adjustment is critical to achieving the uniformity of light on the specimen for photomicrography Most common alignment procedure Aligns the optic lenses and the light source Provides the highest intensity and most uniform illumination for the optical system Uniformity of illumination prevents shadows glare and inadequate contrast 6 Kingdoms Plantae algae plants Animalia Fungi Protista Monera Eubacteria Archaebacteria Kingdom classifications are in flux due to ongoing research in Phylogenomics Intracellular Structures Visible features are limited by the imaging method used Preparing Fresh Mounts Elodea Anacharis water plant Cytoplasmic streaming chloroplasts cell wall Potato tubers Stain w Lugol s Iodine stain Amyloplasts starch granules Onion cells nucleus amp vacuole difficult to see Stain w Lugol s Iodine stain Stomata in fresh leaf mounts Cheek squamous epithelial cells Compare features of premounted tissues Muscle Nerve cells neurons Blood cells Bone Fat adipose tissue Plant epidermis and stomata Compare features of freshmounted cheek cells to those of premounted specimens Cheek squamous epithelial cells Amoeba Algae Spirogyra Oscillotaria or Diatoms Compare cell size and organelles Purple folders extra credit Make observations about one or more images Give complete information about images for which observations are given write complete photo caption in answer OR Make a general comparison of SEM and TEM imaging techniques 10 October Light Microscopes Bright Field Background is brightly lit Objective lenses above specimen Condensor below specimen Phase contrast Special lenses annular stop turret in our lab exercises Background light is separated from the light diffraction pattern generated by the specimen Dark field Background light is dark Opaque disk inserted between light source and condenser Dissecting or stereoscope Large working distance Used for handling large specimens Inverted Objective lens below specimen Condensor above the specimen Used for tissue culture Differential Interference Contrast Separates polarized light into two beams The length of each optical path differs which gives the appearance of a 3D relief Fluorescence Compound or inverted Mercury or xenon light source Mirrors excitation emission filters allow only emitted light of a specific wavelength to be seen Confocal A special type of fluorescence scope Software allows digital capture of image slices Multiple images across the vertical 2 axis are captured and reassembled in 3D reconstruction I In Reduces 39 g from I 39 parts of the specimen Electron Microscopes Transmission Electron Uses high energy electron beam to bombard the specimen 100 nM thick with electrons Dense parts of the specimen do not allow electrons to pass through the specimen Cytoplasmic areas appear clear Resolving Power 3 to 10 Angstroms 3 Scanning electron Used for unsectioned specimens Bombards the surface with electrons to reveal a 3D image Resolving power 30 100 A 1 Angstrom 01 nm 10 A 1 nm Other Microscopes Scanning Tunneling Using a minute probe rather than a high energy Xray beam to scan across a surface Individual atoms of materials can be imaged Nonimaging techniques Atomic Force Microscopy Used to deduce overall shape of single molecules in solution XRay Crystallography Diffraction patterns are used to deduce structure of individual proteins and complexes Atomic Force microscopy Vertical limit of resolution gt 1 Angstrom XRay Crystallography Used to determine subatomic resolution There is more to an objective than just the magnification The numerical aperture determines the quality of the objective Determines the resolving power NA the range of angles over which a system can accept or emit light It is a function of the refractive index of the medium in which the lens works The best light microscopes magnify objects up to 500x what the human eye can see Electron microscopes magnify objects up to 200000x beyond what the human eye can see Adjust the microscope for your use Learn Kohler Illumination Most common alignment procedure Aligns the optic lenses and the light source Provides the highest intensity and most uniform illumination available Uniformity of illumination prevents shadow glare and inadequate contrast 19 September MichaelisMenton Kinetics Describes irreversible catalytic reactions Km MichaelisMenton rate constant Km k2 kcat k1 Reaction rates vary with changes to substrate and enzyme Fast reaction 9 smaller Km Enz Report Plotted Amvs dopachrome mM A Calculate C Generate standard curve y mx b Correlation coef ficient Calc umolesdopachrome D h Plot A475vsumoles 0pamee Used in Enz lab Enz Report Part A Calc Optimum Enzyme req to convert 2 umoles of DOPA to Dopachrome in 3 to 5 minutes 2 mMDopa Cal mirrornoles mm Part B Determined pH effects on Velocity at 15 min Amvs velocity mMDopamicromole Dopachrome umols Plot velocity vs pH min Part C Det Temperature effects on Velocity Amvs Velocity mMDopa micromole Dopachrome umols Plot velocity vs temperature min M VelocIty Is commonly expressed as mm Enz I Report 1 Det A475gt Product mM ampumols In 05 min increments For serial dilution series 5 different Substrate 2 Plot Product Y axis vs Minutes X axis Put all curves on same graph Calc slope m Velocity y2 y1 x1 v1 x2 M M le 3 Determine V for each Dopa concentration 1 1 calc39 5139 05mM 20 1 Calcv Plot Velocity Y axis vs Dopa X axis MichaelisMenton Plot rectangular hyperbola PI tlv 39 ix 39 0 V aXIs versus 5 aXIs LineweaverBurk plot Km Vmax Use LineweaverBurk plot to estimate the Km Vmax and Vmax S Vmax S Vmax 39 KmS 2s 2 and when Km S Vi The LineweaverBurk Equation is Km 1 V1 Vmax S Vmax Protocol Prepare 2 fold dilution series of DOPA in citrate buffer pH 48 Once the enzyme extract has been added each dilution will be used to collect absorbance A475 readings every 30 seconds for 5 minutes BLANK 08 mL of citrate buffer 02 mL enzyme extract 08 mL of8 mM DOPA pH 48 to a disposable plastic cuvette Add 02 mL of enzyme extract Mix Record A475 Designate this reading as time 0 At 30 second intervals read and record the absorbance until a value of 10 is reached Complete the data sheet table Repeat from other Dopa concentrations Three Measures of Enzyme Activity are Commonly Used Turnover number kw number of substrate molecules turned over to product per enzyme molecule per second Vmax Er Kcal Unit U a measure of activity of the enzyme amount of enzyme that produces 1 umole of product per minute Eg 1 U of tyrosinase activity 1 umole of dopachrome produced per minute Specific activity U mg protein 7 November Results of respiration and photosynthesis Mitochondriarespiration Suspension buffer MB 9 ve Control Pellet 700x g MB 9 Chloroplasts Supernatant 700 x g MB 9 Mitochondria ChloroplastsHill Reaction Chloroplasts DCPIP acts as a final electron acceptor gets reduced directly by ETCquantitative assay Mitochondria MB gets reduced indirectly by the depletion of oxygen from the media in the presence of active mitochondrial ETCqualitative assay Fluorescence Microscopy Concepts Detect localize and quantify Proteins molecules and structures Uses flurophores stains fluorescent molecules Can costain multiple structures in the same cell Direct or indirect stains Protocol reasons for steps Types of fluorescence staining Direct staining fluorescent dye fluorochrome directly binds to cellular components Example Dapi and Hoechstnucleic acids Indirect Immunostaining antigenantibody interaction Flurophore conjugated to a probe that binds to specific structures eg Phalloidin Fusion proteins Green fluorescent protein G FP GFP gene is fused to gene of interest allows live cell imaging Fluorescent Molecules Fluorophores Absorb at one WL specific to the fluorophore and emit at another specific wavelength Filter combination blocks Excitation filter filters light wavelengths allowing only specific absorbing wavelength excitation to illuminate the dye in the specimen Emission filter allows only the emitted light emission to reach the eye dye glows against a dark background This week s experiment Fluorescence staining of 3T3L1 fibroblast cells Stain nuclei direct with HoechstBlue Actin filaments PhalloidinAlexafluor488 conjugate Microtubules immunofluorescence 2 ABAlexa Fluor 594 conjugate Steps of staining cells Grow cells on coverslips Fix cells preserves cell structure and antigens STEP C Permeabilize cells Antigen Block with BSA and preimmune serum 1 antibody 2 antibody fluorophore conjugate Hoechst PhalloidinAlexafluor488 Mounting on a slide Controls Do not confuse with the blocking steps Ensure isolated channels Only one dye should be seen in each channel No quotbleedingquot of dye over the other 2 AB alone Preimmune serum from primary antibody Tests for nonspecific binding of primary antibody to proteins in the cell 26 September Part 1 Electrophoresis Greek phoresis transmission Gel electrophoresis A technique of molecular separation The movement of charged molecules in an electric field Molecules separate in a porous matrix gel according to their charge size and shape Proteins are charged molecules Proteins are chains of amino acids Types of protein separation 1 Native gel electrophoresis proteins separate by size charge and conformation Uses isozymes enzyme activity 2 soelectric focusing proteins separate by their isoelectric point pl in a pH gradient p the pH at which protein net charge is zero 3 SDS Denatured Gels separate proteins according to size MW Boiling breaks Hbonds SDS breaks noncovalent bonds coats with ve charge BMercatoethanol 55 9 SH Migration of denature proteins inversely proportional to their MW 4 2D gels 2 amp 3 High resolution separation Used in proteomics analyses Types of gels Tube gels Slab gels Continuous gels Discontinuous gels Tquot 39 gel 39 r39 39 system F I Resolving gel 12 polyacrylamide Stacking get 4 polyacrylamide Lower electrode buffer lower chamber Upper electrode buffer upper chamber Protein sample Anode amp cathode Part 1 Gel Electrophoresis Separation of partially purified potato tyrosinase on SDS denaturing gel electrophoresis 2X Sample Buffer Buffer TrisHCI pH 68 Glycerol 10 SDS detergent Bromophenol blue BMercaptoethanol Why Glycerol Keeps it in wells Makes it heavy Why SDS Opens tertiary structure of proteins Four factors are responsible for tertiary structure Disulfide linkages Hydrogen bonding Electrostatic interactions Hydrophobic interactions Breaks hydrophobic interactions adds ve charge Why BMercaptoethanol 55 9 SH Why bromophenol blue Can see that it s loaded in wells Gel Electrophoresis Determining Protein Molecular Weights MW A standard curve is made relating the logarithm of standard protein MW size markers yaxis with the distance they migrate on the gel This curve is used to determine the size MW of unknown proteins by measuring the distance they migrate on the gel and the line of best fit Part 2 Protein Quantification Spectrophotometer uV Absorption Aromatic amino acids Colorimetric visible wave length Dyes Bradford reagent Coomassie Blue 6250 Arginine some aromatic BCA Method Bicinchoninic acid copper in an alkaline medium proteincopper chelate that has a distinct blue color A562 nm Fluorescence Gel electrophoresis Choice Based upon interfering agents eg detergents pH availability of instruments Part 2 Protein Quantification Bradford Assay Bradford reagent maximum absorbance Lmax is A495 red proteins Lmax is 9 A595 blue Absorbance of Bradfordprotein complex at 595 nm is proportional to the concentration of proteins Beer s Law applies if the protein is pure Bradford Assay BSA Standard curve Start measuring from low to high conc Dispose of Bradford reagent in hazardous waste Pipette standards consistently Why Standard Curve Mixture of proteins Extinction coefficient is not known May not follow Beer s Law Tips for protein gel electrophoresis Wear gloves Polyacrylamide is neurotoxic in its monomer form Dispose of polymerized gels in the trash Bromophenol Blue BB is also moderately hazardous Bmercaptoethanol has a very unpleasant smell in addition to its toxicity if inhaled use the hood for adding sample buffer and loading your gel Sample buffer is viscous pipette in and out slowly to prevent inaccurate measurement Assembling the electrophoresis gels and tank Leakage notch of gasket against the smaller glass plate Boiling the samples watch for popping of tube caps Steady hand and avoid introducing bubbles into your wells practice gel 22 August LIFE 212 course information Syllabus read the syllabus thoroughly Laboratory Manual 2nd edition RamCT Lectures Updates Discussions TA Office hours spread over the week Lecture 30 40 min in Recitation powerpoints Quiz Quizzes Prelabwriteup Lab notebooksverified bysign out with TA Lab reports Due on Mondays in Recitation Lab tech grade 10 points Absence not allowed Extra credit questions Lab notebook writeup Introduction Materials and methods Results and discussion tear out report Fill submit for grading tape the graded report in your notebook Conclusion Use ink pens for writing Cross out any empty spaces LIFE 212 Course Information Prelab writeup Introduction materials and protocol Verified bysign in and out with TA After the lab Fill the Report turn in for grading Tape the gm report in the notebook Write conclusions see syllabus Quizzes Open notebook Includes material from last and current week s lab Lab reports Due on Mondays in Recitation one week after the lab Late assignments lose points Lab tech grade 10 points Prelabwriteup 5 points Bench cleanup glassware return and seriousness about conducting the experiments 5 points Absence Missing a lab is NOT allowed Contact instructors ONLY Prior arrangement to attend other sections during the week Illness needs doctor s note LIFE 212 Testing and Grading Assessment Pts each Total points 11 Quizzes 20 220 13 13 Lab Reports 50 100 760 44 2 Notebook grades 100 200 12 2 Exams 200 400 234 13 Lab tech grade 10 130 76 Total 1710 points 100 EXTRA CREDIT QUESTIONS are not part of the total points of 1710 Student Misconduct Cheating and Plagiarism are not allowed and can result in serious disciplinary actions Experiment 1 Objectives Part 1 Express concentrations of solutions Convert concentrations from one expression to another Part 2 Accuracy Precision of data measurement Part 1 concentrations of solutions Units Concentrations of solutions Units Biochemists use units such as grams moles and Liters Their multiples and subdivisions Milli 10393 Micro 10396 Nano 10 9 Pico 103912 Write leginy Concentrations of Solutions Expressions and calculations Solution solute dissolved in solvent Amount quantity grams moles or vs volume The amount of solute Concentration The Volume of solvent t l ammm g or me Concentration Volume L L m mol mmol mmol Examples g g H or or or L mL mL L L mL Three Expressions of Concentrations Molarity mol T 39V39 Mass mg Volume L39 E g mL Percentage 100 mL or 100 mL Conversions g mol and M l BasIc equations to convertto M and VIce versa MW L mol mol M T Percentage denominator 100 mL Does not require formula wt MW 0 K I grams Weight to volume A 17100 mL Example a 10 solution has 10 g of solute in a total of 100 mL of solution 0 Z I mL Volume to volume A 17 100 mL Example a 10 E solution of ethanol has 10 mL of pure ethanol in 100 mL of total solution Dilutions Dilutions are made from stock solutions to meet protocol needs Dilution factor DF Dilution is made of the whole parts 15 5 parts in which one is a part of 5 The number after 1 is the dilution factor 1250 Dilution factor 250 11000 Dilution factor 1000 Basic Equation C1V1 C2V2 C1 initial conc C2 final conc V2 final volume Vl initial volume V2 Vl Or a C2 DF DF 52 V2 DF Example to make 500 mLof 1M from a solution of 5M you use the formula C1V1 C2V2 C1 5M C2 1M V2 500 mL Vl Vl 100 mL 1M500 mL 5 Serial dilutions Basic equations FinalDFDF1DF2DF3etc Data 2 Data Measurement Objectives Learn the concept of accuracy and precision in measurement Measure volumes using the micropipettes Accuracy s how close a measurement is to the true value of the property being measured A measurement is accurate if it gives the true value Depends on the design of the measuring device I 0 Accurac Is ex ressed as V error experimental Valu eitrue Value error x 100 true m1 lue Acceptable values are S 05 1 Precision I Measures the r I of your Expressed as mean deviation from the mean Mean of absolute deviations of individual measurement from the mean value Example M1 1498 g M2 1502 g M3 1497 g Mean 1499 g 149815021497 3 Deviation for m1 1498 1499I 001 Deviation for m2 1502 1499I 003 Deviation for m3 1497 1499I 002 001003002 002 g Final reported value for the mass 1499 g i 002 g Acceptable values are S 05 1 of the mean Note the units are always shown on a reported measurement Volume measuring devices Graduated cylinders volumetric pipettes Do not use the graduations of beakers or flasks those are not accurate Micropipettes Pipetman P1000 P200 P20 P10 P2 Pipette use rules Pipettes are accurate to 110 of their volume Example 1 mL is accurate to 100 uL P200 is accurate to 20 uL P20 is accurate to 2 uL P10 is accurate to 1 uL Use the smallest pipetman that contains the volume you need to measure
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