Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-47 (Electronic) How to Properly Use and Maintain Laboratory Equipment Trevor Troutman Kristin A. Prasauckas Michele A Kennedy, Jane Stevens, Michael G. Davies, and Andrew T Dadd Balances and scales 5 How Are Balances and scales Characterized? How Can the Characteristics of a Sample and the Immediate Environment Affect Weighing Reproducibility? 5 By What Criteria Could You Select a Weighing Instrument? How Can You generate the most reliable and Reproducible Measurements? How Can You minimize service calls 55 Centrifugation Theory and Strategy Practice Centrifugation of DNA and rNa 63 Troubleshooting Pipettor Data on the performance characteristics of different protein concentration assays were generously provided by Bio Rad Inc
49 4 How to Properly Use and Maintain Laboratory Equipment Trevor Troutman, Kristin A. Prasauckas, Michele A. Kennedy, Jane Stevens, Michael G. Davies, and Andrew T. Dadd Balances and Scales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 How Are Balances and Scales Characterized? . . . . . . . . . . . . 51 How Can the Characteristics of a Sample and the Immediate Environment Affect Weighing Reproducibility? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 By What Criteria Could You Select a Weighing Instrument? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 How Can You Generate the Most Reliable and Reproducible Measurements? . . . . . . . . . . . . . . . . . . . . . . . 54 How Can You Minimize Service Calls? . . . . . . . . . . . . . . . . . . 55 Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Theory and Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Centrifugation of DNA and RNA . . . . . . . . . . . . . . . . . . . . . . 63 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 Pipettors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Data on the performance characteristics of different protein concentration assays were generously provided by Bio Rad Inc. Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)
Which Pipette Is Most Appropriate for Your Application? What Are the Elements of Proper Pipetting Technique? 68 Preventing and Solving Problems Troubleshooting PH Meters What Are the Components of a pH Meter? 8777 How Does a pH Meter Function? How Does the Meter Measure the Sample PH? What ls the Purpose of Autobuffer Recognition? Which Buffers Are Appropriate for Your Calibration What ls Temperature Compensation and How Does One Choose the Best Method for an Analysis?........ 84 How Does Resolution Affect pH Measurement? Why Does the Meter Indicate"Ready"Even as the H Value Changes Which PH Electrode Is Most Appropria te for your Analysis? How Can You Maximize the Accuracy and Reproducibility of a ph Measurement? How Do Lab measurements differ from plant or field Does Sample Volume Affect the Accuracy of the PH How Do You Measure the PH of Viscous, Semisolid, Low lonic Strength, or Other Atypical Samples How Can You Maximize the Lifetime of Your pH Meter? Troubleshooting Is the instrument the problem? 92 Service Engineer, Technical Support, or Sales Rep: Who Can Best Help You and at the Least Expens Spectrophotometers What Are the Criteria for Selecting a Spectrophotometer? Beyond the Self-Tests Automatically Performed by Spectrophotomters, What Is the Best Indicator That an Instrument Is Operating Properly Which Cuvette best fits your needs? What Are the Options for Cleaning Cuvettes? How Can You Maximize the Reproducibility and Accuracy f Your data? What Can Contribute to Inaccurate A260 and a280 Data? Troutman et al
Which Pipette Is Most Appropriate for Your Application? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 What Are the Elements of Proper Pipetting Technique? . . . 68 Preventing and Solving Problems . . . . . . . . . . . . . . . . . . . . . . . 68 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 pH Meters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 What Are the Components of a pH Meter? . . . . . . . . . . . . . 77 How Does a pH Meter Function? . . . . . . . . . . . . . . . . . . . . . . 80 How Does the Meter Measure the Sample pH? . . . . . . . . . 81 What Is the Purpose of Autobuffer Recognition? . . . . . . . . . 82 Which Buffers Are Appropriate for Your Calibration Step? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 What Is Temperature Compensation and How Does One Choose the Best Method for an Analysis? . . . . . . . . 84 How Does Resolution Affect pH Measurement? . . . . . . . . . 85 Why Does the Meter Indicate “Ready” Even as the pH Value Changes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Which pH Electrode Is Most Appropriate for Your Analysis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 How Can You Maximize the Accuracy and Reproducibility of a pH Measurement? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 How Do Lab Measurements Differ from Plant or Field Measurements? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 Does Sample Volume Affect the Accuracy of the pH Measurement? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 How Do You Measure the pH of Viscous, Semisolid, Low Ionic Strength, or Other Atypical Samples? . . . . . . . . . . . . 90 How Can You Maximize the Lifetime of Your pH Meter? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 Is the Instrument the Problem? . . . . . . . . . . . . . . . . . . . . . . . 92 Service Engineer, Technical Support, or Sales Rep: Who Can Best Help You and at the Least Expense? . . . . . . . . . 94 Spectrophotometers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 What Are the Criteria for Selecting a Spectrophotometer? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 Beyond the Self-Tests Automatically Performed by Spectrophotomters, What Is the Best Indicator That an Instrument Is Operating Properly? . . . . . . . . . . . . . . . . 98 Which Cuvette Best Fits Your Needs? . . . . . . . . . . . . . . . . . . 100 What Are the Options for Cleaning Cuvettes? . . . . . . . . . . 101 How Can You Maximize the Reproducibility and Accuracy of Your Data? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 What Can Contribute to Inaccurate A260 and A280 Data? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 50 Troutman et al
Does Absorbance Always Correlate with Concentration Why Does Popular Convention Recommend Working Between an absorbance range of 0. l to 0.8 at 260 nm When Quantitating Nucleic Acids and When Quantitating Proteins at 280 nm Is the Ratio, A260: A280, a Reliable Method to Evaluate Protein Contamination within Nucleic Acid Pr What Can You do to minimize service calls? How Can You achieve the maximum Lifetime from Your lamps The Deuterium Lamp on Your UV-Visible Instrument Burned out. Can You perform measurements in the Visible Range? 107 What Are the strategies to determine the Extinction Coefficient of a Compound? What Is the extinction coefficient of an Oligonucleotide? Is There a Single Conversion Factor to Convert Protein bsorbance data into concentration? What Are the Strengths and Limitations of the various Protein Quantitation Assays? 09 Bibliography BALANCES AND SCALES (Trevor Troutman) How Are Balances and scales Characterized? Balances are classified into several categories. Top-loaders are balances with 0.001 g or 1 mg readability and above, where readability is the lowest possible digit that is seen on the display Analytical balances are instruments that read 0. 1mg. Semimicro balances are those that are 0.01 mg. Microbalances are l ug Finally, the ultramicrobalance are 0. 1 ug How Can the Characteristics of a Sample and the Immediate Environment Affect Weighing Reproducibility? Moisture Condensation forms on reagents that are not kept airtight while they equilibrate to room temperature. Similarly moisture generated if the sample is not allowed to reach the temperature of the weighing instrument. This is especially problematic when weighing very small samples. You the researcher are also a source of moisture that can be transmitted quite easily to the sample in the form of fingerprints and body oils. How to Properly Use and Maintain Laboratory Equipment 5
Does Absorbance Always Correlate with Concentration? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 Why Does Popular Convention Recommend Working Between an Absorbance Range of 0.1 to 0.8 at 260nm When Quantitating Nucleic Acids and When Quantitating Proteins at 280nm . . . . . . . . . . . . . . . . . . . . 105 Is the Ratio, A260 :A280, a Reliable Method to Evaluate Protein Contamination within Nucleic Acid Preparations? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 What Can You Do to Minimize Service Calls? . . . . . . . . . . . 106 How Can You Achieve the Maximum Lifetime from Your Lamps? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 The Deuterium Lamp on Your UV-Visible Instrument Burned Out. Can You Perform Measurements in the Visible Range? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 What Are the Strategies to Determine the Extinction Coefficient of a Compound? . . . . . . . . . . . . . . . . . . . . . . . . 108 What Is the Extinction Coefficient of an Oligonucleotide? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Is There a Single Conversion Factor to Convert Protein Absorbance Data into Concentration? . . . . . . . . . . . . . . . . 108 What Are the Strengths and Limitations of the Various Protein Quantitation Assays? . . . . . . . . . . . . . . . . . . . . . . . . 109 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 BALANCES AND SCALES (Trevor Troutman) How Are Balances and Scales Characterized? Balances are classified into several categories. Top-loaders are balances with 0.001 g or 1 mg readability and above, where readability is the lowest possible digit that is seen on the display. Analytical balances are instruments that read 0.1 mg. Semimicro balances are those that are 0.01mg. Microbalances are 1mg. Finally, the ultramicrobalances are 0.1mg. How Can the Characteristics of a Sample and the Immediate Environment Affect Weighing Reproducibility? Moisture Condensation forms on reagents that are not kept airtight while they equilibrate to room temperature. Similarly moisture is generated if the sample is not allowed to reach the temperature of the weighing instrument. This is especially problematic when weighing very small samples. You the researcher are also a source of moisture that can be transmitted quite easily to the sample in the form of fingerprints and body oils. How to Properly Use and Maintain Laboratory Equipment 51
Air Buoy Akin to water keeping something afloat, samples can be"lifted by air, artificially decreasing their apparent weight. This air buoy- ancy can have a significant effect on smaller samples. Electrostatic forces Electrostatic charges are almost always present in any enviro ment, particularly in areas with very low humidity. If there are considerable charges present in a sample to be weighed on a high precision instrument, it will manifest itself in the form of drifting constant increase or decrease of weight readings, or nonrepro- ducible results. Variability occurs when these electrical forces build up on the sample and the fixed parts of the balance that are not connected to the weighing pan Substances with low electrical conductivity (e. g, glass, plastics, filter materials, and certain powders and liquids) lose these charges slowly, prolonging the drift during weighing. The charges most likely originate when the sample is being transported or processed. Examples include friction with air in a convection oven, friction between filters and the surface they contact, internal friction between powders and liquids during transportation, and direct transfer of charged particles by persons. This charge accumulation is best prevented by use of a Faraday cage, which entails shielding a space in metallic walls. This frees the inside area from electrostatic fields. a metallic item can serve the same purpose. Surrounding the container that houses the reagent in foil can also reduce charge accumulation. For nonhy groscopic samples, adding water to increase the humidity inside the draft chamber can reduce static electricity. accomplish this by placing a beaker with as much water as possible into the draft chamber. An alternative is to bombard the sample with ions of the opposite charge, as generated by expensive ionizing blowers and polonium radiators. A simple and effective solution is to place an inverted beaker onto the weigh pan, and then place the sample to be weighed onto the inverted beaker. This strategy increases the distance between the sample and the weigh pan, thus weakening any charge effects. Tempere Airing out a laboratory or turning the heat on for the first time with the change of seasons has a profound effect on an analytical balance. The components of a weighing system are of different size and material composition, and adapt to temperature changes at Troutman et al
Air Buoyancy Akin to water keeping something afloat, samples can be “lifted” by air, artificially decreasing their apparent weight. This air buoyancy can have a significant effect on smaller samples. Electrostatic Forces Electrostatic charges are almost always present in any environment, particularly in areas with very low humidity. If there are considerable charges present in a sample to be weighed on a highprecision instrument, it will manifest itself in the form of drifting, constant increase or decrease of weight readings, or nonreproducible results. Variability occurs when these electrical forces build up on the sample and the fixed parts of the balance that are not connected to the weighing pan. Substances with low electrical conductivity (e.g., glass, plastics, filter materials, and certain powders and liquids) lose these charges slowly, prolonging the drift during weighing. The charges most likely originate when the sample is being transported or processed. Examples include friction with air in a convection oven, friction between filters and the surface they contact, internal friction between powders and liquids during transportation, and direct transfer of charged particles by persons. This charge accumulation is best prevented by use of a Faraday cage, which entails shielding a space in metallic walls. This frees the inside area from electrostatic fields. A metallic item can serve the same purpose. Surrounding the container that houses the reagent in foil can also reduce charge accumulation. For nonhygroscopic samples, adding water to increase the humidity inside the draft chamber can reduce static electricity. Accomplish this by placing a beaker with as much water as possible into the draft chamber.An alternative is to bombard the sample with ions of the opposite charge, as generated by expensive ionizing blowers and polonium radiators. A simple and effective solution is to place an inverted beaker onto the weigh pan, and then place the sample to be weighed onto the inverted beaker. This strategy increases the distance between the sample and the weigh pan, thus weakening any charge effects. Temperature Airing out a laboratory or turning the heat on for the first time with the change of seasons has a profound effect on an analytical balance.The components of a weighing system are of different size and material composition, and adapt to temperature changes at 52 Troutman et al
different rates. When weighing a sample, this variable response to temperature produces unreliable data. It is recommended to keep a constant temperature at all times in an environment where weighing instruments are kept. When room temperature changes, allow the instrument to equilibrate for 24 hours. Air Currents or Drafts The flow rate of ambient air should be minimized to get nd stable results with weighing equipment For balances with a readability of 1 mg, an open draft shield (glass cylinder) will suffice. Below 0. 1 mg, a closed draft chamber is needed These shields or chambers should be as small ible eliminate convection currents within the chamber to minimize temperature variation and internal draft problems. Magnetic Forces and Magnetic Sample Magnetic forces are produced when a sample is magnetized or magnetizable, which means it contains a percentage of iron, cobalt or nickel. Magnetic effects manifest themselves in the sample's loss of reproducibility. But unlike electrostatic forces, magnetic forces can yield a stable measurement. Changing the orientation of the magnetic field(moving the reagent sample) relative to the weigh system causes the irreproducible results magnetic effects are thus difficult to detect unless the same sample is weighed more than once. Placing an inverted beaker or a piece of wood between the sample and the pan can counteract the magnetic force. Some instruments allow for below balance weighing, in which a hook used to attach magnetic samples lies underneath the weigh pan at a safe distance in order to eliminate magnetic effects. Gravitational tilt a balance must be level when performing measurements on the weighing pan. Gravity operates in a direction that points straight to the center of the earth. Thus, if the weigh cell in not directly in this path, the weight will end up somewhat less. For example, say we weigh a 200g sample that is 0. 2865(angle a) out of paral lel. we have Apparent weight weight * cos a Apparent weight = 200*cos 0. 2865=199.9975g This result represents a 2.5mg deviation. This is a significant quantity when working with analytical samples How to Properly Use and Maintain Laboratory Equipment 53
different rates. When weighing a sample, this variable response to temperature produces unreliable data. It is recommended to keep a constant temperature at all times in an environment where weighing instruments are kept. When room temperature changes, allow the instrument to equilibrate for 24 hours. Air Currents or Drafts The flow rate of ambient air should be minimized to get quick and stable results with weighing equipment. For balances with a readability of 1 mg, an open draft shield (glass cylinder) will suffice. Below 0.1 mg, a closed draft chamber is needed. These shields or chambers should be as small as possible to eliminate convection currents within the chamber to minimize temperature variation and internal draft problems. Magnetic Forces and Magnetic Samples Magnetic forces are produced when a sample is magnetized or magnetizable, which means it contains a percentage of iron, cobalt, or nickel. Magnetic effects manifest themselves in the sample’s loss of reproducibility. But unlike electrostatic forces, magnetic forces can yield a stable measurement. Changing the orientation of the magnetic field (moving the reagent sample) relative to the weigh system causes the irreproducible results. Magnetic effects are thus difficult to detect unless the same sample is weighed more than once. Placing an inverted beaker or a piece of wood between the sample and the pan can counteract the magnetic force. Some instruments allow for below balance weighing, in which a hook used to attach magnetic samples lies underneath the weigh pan at a safe distance in order to eliminate magnetic effects. Gravitational Tilt A balance must be level when performing measurements on the weighing pan. Gravity operates in a direction that points straight to the center of the earth. Thus, if the weigh cell in not directly in this path, the weight will end up somewhat less. For example, say we weigh a 200 g sample that is 0.2865° (angle = a) out of parallel. We have Apparent weight = weight * cosa Apparent weight = 200 *cos0.2865 = 199.9975 g This result represents a 2.5mg deviation. This is a significant quantity when working with analytical samples. How to Properly Use and Maintain Laboratory Equipment 53