QBM 3

Lecture 3: Micropipettes, Centrifuges, and Spectrophotometers

Lecture Overview

  • Laboratory Basics

  • Transferring Liquids

  • Micropipettes

  • Centrifugation

  • Spectroscopy and Spectrophotometry

I. Laboratory Basics

  • Laboratory Research Notebook

    • Department

    • Subject

    • Name

    • Address

Micropipetting Protocol

  • Each partner to perform a single micropipetting protocol individually and then in triplicate (three replicates).

  • Experimental data will be compared, graphed, and analyzed afterward.

Micropipetting Individual Protocol

  1. Obtain six 1.5 ml microcentrifuge tubes and label as follows:

    • One tube labeled Blank

    • Five tubes labeled 1 through 5

  2. Using Table 1-1, add indicated volumes of solutions to each tube. All measurements of Solution A can be done using the same pipette tip.

    Tube #

    Solution A (μl)

    Solution B (μl)

    Solution C (μl)

    Solution D (μl)

    Blank

    800

    150

    50

    2

    1

    796

    150

    50

    4

    2

    794

    150

    50

    6

    3

    792

    150

    50

    8

  3. Add 150 μl of Solution B to each tube using the same tip as long as it does not contact any Solution A. If immersion occurs, change the tip. Do not cap any tubes.

Glassware Cleaning and Sterilization

  • Clean glassware with soap or Alconox, scrub well with a brush.

  • Rinse thoroughly:

    • Hot water (approximately 3 times)

    • Deionized water (approximately 3 times)

  • Rinsing prevents any contamination from detergent or acetone, which can affect biological samples used in molecular biology.

  • Sterilization options:

    • Sterilization of glassware and media using an autoclave.

    • Decontamination Techniques:

    • Use ethanol for benches

    • Use bleach for cells

II. Transferring Liquids

  • Transferring liquids is fundamental in molecular/biochemical experiments; accurate and consistent transfers yield accurate results.

  • Recommended tools based on volume ranges:

    • Greater than 2 L: Use graduated cylinder, beaker.

    • 25 ml to 2 L: Use graduated cylinder.

    • 1 to 25 ml: Use glass or plastic pipettes.

    • 0.0001 (1/10th ml) to 1 ml: Use micropipettes.

Volumetric Measurements Types

  • Burettes

  • Pipets

  • Volumetric Flasks

  • Graduated Cylinders

Glass/Plastic Pipettes Types

  • Mohr Pipette: Designed to contain; not to blow out, with a dead space below last graduation (> 1 ml). Accurate for multiple dispenses from a single aspiration.

  • Serological Pipette: Designed to deliver; must blow out for accuracy, filled to a specific volume.

  • Volumetric Pipettes: Designed to deliver a specific volume.

  • Transfer (Pasteur) Pipettes: Generally uncalibrated and disposable, used when precision is not a priority.

Pipette Aids

  • Pipette Delivery Aids:

    • Pipette fillers

    • Rubber pumps

    • Motorized pipettes

    • Transfer pipettes

Micropipette Features

  • Parts of a Micropipette:

    • Plunger

    • Tip ejector

    • Volume adjustment

    • Volume indicator

    • Disposable plastic tip

III. Micropipette Usage

  • Select a micropipette that matches the approximate volume needed (e.g., use a P10 for 5 μL).

  • Ensure the tip is tightly fitted over the aspirating end.

  • Procedures:

    1. Press down to the first stop with your thumb.

    2. Insert the tip into the liquid.

    3. Slowly release the plunger to draw liquid.

    4. Place the tip in the receiving tube.

    5. Push the plunger to the second stop to dispense the liquid.

    6. Remove the tip and release the plunger.

Sources of Error in Micropipetting

  • Common Errors:

    • Uncalibrated micropipettor

    • Dirt or debris in the micropipettor

    • Liquid on the tip's exterior

    • Incomplete mixing

    • Improper pipetting techniques

    • Both systematic and random errors

Micropipette Care

  • Micropipettes must be handled with care and need calibration yearly.

  • Do Not:

    • Rotate the volume adjuster beyond limits

    • Use without a tip (fluid can damage the piston)

    • Immerse the barrel in fluids

    • Hold horizontally with a filled tip (risk of backflow)

    • Snap back control button after withdrawing fluid

    • Flame a micropipette tip

    • Jam the pipette into the holder (can bend the metal tube)

Handheld Pipetting Best Practices

  • Pre-Wet Tips: Aspirate/dispense nominal volume three times after loading tips to equilibrate temperature differences and humify the air space.

  • Temperature Equilibrium: Ensure the pipette, tips, and liquids are at room temperature to prevent volume inconsistencies.

  • Consistent Pipetting Angle: Maintain a pipette angle not exceeding 20 degrees to ensure consistent aspiration volumes.

Aspirating Techniques

  • Aspirate Just Below Surface: Immerse 2-3 mm below the surface of the liquid to prevent droplets clinging to the outside of the tip.

  • Optimize Volume Range: Use within 35%-100% of nominal volume for best accuracy; avoid low-volume ranges.

  • Discard First/Last Dispense: First and last dispenses in multiple aliquots tend to have the highest error and should be discarded.

  • Touch Off: Use the side wall touch-off for accuracy when dispensing directly into a vessel.

Specific Conditions in Pipetting

  • Pipetting Viscous Liquids: Aspirate and dispense slowly or use ‘Reverse pipet’ mode to compensate for retained liquid.

  • Pipetting Volatile Liquids: Pre-wet tips and use ‘Reverse pipet’ mode to minimize evaporation.

  • Calibration Based on Liquid Density: Recalibrate if using liquids with different densities than water for accurate dispensing.

IV. Centrifuge

  • The centrifuge was first used in the mid-1800s to separate milk, reaching speeds of 3000 rpm.

  • Modern Centrifuges: Can reach 150,000 rpm and up to 1,000,000 g.

  • Developed by Theodore Svedberg in the early 1900s, who conducted analytical ultracentrifugation to measure hemoglobin weight.

    • 1 Svedberg = 100 femtoseconds (10^{-13} seconds).

Purpose of Centrifugation

  • Main Functions:

    • Separate samples based on mass, shape, and density.

    • Commonly used to separate cells from media, insoluable fractions after bacterial lysis, types of organelles, and more.

    • Concentrate biological substances like DNA, RNA, and proteins.

Process Description

  • Spin Down Procedure: Involves inverting/mixing a sample followed by a spin-down to gather fluid into a cohesive solution.

  • RPM and g-forces can vary based on the type of centrifugation performed, impacting the sedimentation process significantly.

Separation by Sedimentation

  • Particles naturally sediment out of solutions; centrifuges enhance sedimentation through increased applied forces.

  • The sedimentation rate is determined by the centrifugal field g, related to:

    • Angular velocity of the rotor (in RPM),

    • Radius from the rotation axis (r, in cm).

    • Various rotor types and designs also affect sedimentation.

    • RCF (Relative Centrifugal Force) is often expressed as multiples of Earth's gravity, where 1 g = 9.81 m/s².

RPM and RCF Calculations

  • Equation for g-force:
    RCF=11.2imesrimesrac(RPM/1000)21000RCF = 11.2 imes r imes rac{(RPM/1000)^2}{1000}

  • Example:

    • Rotor A Type: 14,000 rpm, 5.98 cm radius, resulting in 13,100 g.

    • Rotor B Type: 14,000 rpm, 9.50 cm radius, resulting in 20,817 g.

Interpreting Protocols

  • Proper protocol interpretation is vital and must include:

    • Specific rotor types, RPM, RCF, and temperature conditions.

  • Example Protocol:

    • Ultracentrifuge G-actin for 1 hour at 4 °C at 100,000 g; vary depending on rotor designs.

Types of Centrifugation

  1. Preparative:

    • Differential and small bench centrifugation, as well as large and high-speed centrifuges.

  2. Analytical:

    • Utilizes density gradients and specially designed rotors for observing molecular sedimentation.

Preparative Centrifugation

  • Most common form of centrifugation useful for practical applications:

    • Separation, isolation, and purification involving large media volumes with bacteria, viruses, and nucleic acids.

  • Analyzes attributes like morphology and biological activity.

Analytical Centrifugation

  • Focuses on examining pure biological macromolecules and their sedimentation characteristics using less material and specialized rotors.

  • Measures purity, molecular weight/size, and other key molecular data.

Centrifuge Types

  • Small Bench Centrifuges:

    • Used for small sample volumes, such as 1 ml microcentrifuge tubes.

    • Typical RPM ~ 13,000, yielding ~ 7,000 g force.

  • Large Refrigerated Centrifuges:

    • Capacities of up to 1 L bottles with RPM around 6,000-6,500 suitable for erythrocytes.

  • High-Speed Refrigerated Centrifuges:

    • Operate at ~25,000 rpm (~60,000 g), focusing on cellular debris and organelles.

  • Ultracentrifuges:

    • Highest performance at ~150,000 rpm (~1,000,000 g) for isolating organelles, viruses, and nucleic acids.

    • Safety features, including temperature sensors and armor plating for protection.

  • Continuous Flow Centrifuges:

    • Designed for harvesting large volumes of cultures by allowing particles to separate within a moving rotor.

Types of Rotors

  • Fixed-Angle: Ideal for pelleting.

  • Swinging-Bucket: Best for size/density purification.

  • Zonal: Suitable for separating large volumes.

  • Vertical: Often utilized for DNA and virus purification.

V. Spectroscopy and Spectrophotometry

  • Definitions:

    • Spectroscopy: The study of light interaction with molecular structures.

    • Spectrometry: The measurement of this interaction generating spectra to analyze the material's characteristics quantitatively and qualitatively.

Categories of Spectroscopy

  • Electromagnetic Spectroscopy Types:

    • Visible, UV, infrared (IR), fluorescence, atomic absorption, X-ray, and nuclear magnetic resonance (NMR).

  • Non-electromagnetic Spectroscopy Types:

    • Mass spectroscopy, electron spectroscopy, and acoustic spectroscopy.

Infrared Spectroscopy (IR)

  • Operates within a wavelength region of ~1000-200,000 nm, analyzing energy transitions associated with molecular vibrations.

  • Utilizes techniques such as Fourier Transform Infrared (FTIR) for various studies, including membrane analyses.

Light Absorbance

  • Every substance in solution has a characteristic absorbance profile, akin to physical properties such as melting point.

  • Absorbance can be related to the concentration of a given substance in solution, thereby serving as a quantitative measurement tool.

Spectrophotometry

  • An analytical technique for determining the concentration and characteristics of a substance by measuring light absorption at specific wavelengths.

  • Spectrophotometry differs from colorimetry, where samples undergo transformations to colored compounds.

  • The relationship between absorbance and concentration adheres to Beer-Lambert law when the path length remains constant.

Spectrophotometer Functionality

  • Measures light intensity across a range, comparing transmitted light through a blank and a sample.

    • Components:

      • Light Source: Tungsten lamp for visible light, deuterium for UV.

      • Monochromator: Selects a wavelength for measurement.

      • Prism/Grating: Disperses light, with exit slit narrowing wavelength selection.

      • Sample Compartment: Holds cuvettes for light exposure to the detector.

      • Detector: Converts light into an electrical current for analysis.

Plate Readers

  • Designed to evaluate multiple samples concurrently, typically in 96-well plates, measuring absorbance, fluorescence, and luminescence across small volumes (5-200 μl).

  • Applications include various assays related to DNA, RNA, proteins, and drug screenings.