Molecular Biology Lab: Automatic Pipettes Guide

• Exercises cover organization, course credit rules, and lab safety/health rules.
• Students learn about basic research devices used in molecular biology labs.

Automatic Pipettes

• Utilized for quickly measuring small liquid volumes, usually under 1 ml.
• Contains a piston, spring, handle, regulator, and replaceable tip.
• Tips typically consist of Teflon or polyethylene.
• Liquid is drawn into the tip using a plunger mechanism.
• The regulator limits the piston's stroke, with a volume scale knob for adjustment.
• Adjustment range is indicated on the intake button with color-coding.
• Available in fixed-capacity or multi-channel versions; some models have automatic tip removal.

Intake Button Colors and Volume Ranges

• Blue: 100 to 1000 μl
• Yellow: 10 to 100 μl
• Red: 0.5 to 10 μl

Accurate Pipetting Tips

• Rinse the tip with the liquid to be pipetted before use, especially for viscous or dense liquids.
• Hold the pipette upright, ensuring the tip is at a near-vertical angle (under 20 degrees) in the sample.
• Pipette position is crucial for dispensing volumes under 50 μl.
• Touching the container sides can cause volume loss due to surface tension.
• Immerse large-volume pipette tips (1-5 ml) to 5-6 mm depth.
• Immerse smaller-volume pipette tips to 2-3 mm depth.
• Avoid too little immersion, which can cause air suction, and excessive immersion, which can cause sticking.
• Do not touch the container bottom with the tip.
• Operate the pipetting button slowly and smoothly.
• Rapid liquid withdrawal can flood the pipette shaft and cause air bubbles.
• Consistent button pressure and speed ensure repeatable results.
• Choose a pipette with a volume range slightly above the desired volume for better accuracy.

General Pipetting Guidelines

• Replace the tip with each new liquid.
• Ensure no liquid remains in the tip from previous tests before each experiment.
• Maintain an even pipetting rate and avoid rushing.

Things to Avoid When Pipetting

• Setting volumes beyond the pipette's adjustment scale.
• Using a pipette without a properly attached tip.
• Laying down a pipette with a filled tip, as it can flood the piston seal.
• Abruptly releasing the plunger while filling the tip.
• Immersing the pipette body in the dispensing liquid.

Pipetting Reminders

• Pipette liquids that may foam slowly.
• Avoid pushing the plunger to the second resistance to prevent air bubbles and foaming.
• Pipette viscous solutions slowly for accuracy.
• When pipetting volatile liquids, pre-saturate the pipette with solution vapors to prevent "leaking".
• Do not walk around the lab with a filled pipette.

Pipetting Techniques

• Standard pipetting (straight pipetting) is used for aqueous solutions like diluted acids, bases, and buffers.
• Reverse pipetting (differential pipetting) is used for thick, viscous, or foaming liquids and small volumes.

Practical Skills Gained

• Operating automatic pipettes for various liquids.
• Using centrifuges, vortex mixers, and electronic analytical balances.

Exercise 1: Preparing Colored Sucrose Solution

• Pipette 405 µl of sucrose solution into a 1.5 ml tube.
• Add 95 µl of dye on top.
• Observe phase boundaries.
• Mix using the pipette and vortex.
• Centrifuge.
• Divide into two tubes.
• Note observations.

Exercise 2: Adding Small Volumes

• Prepare a 1.5 ml tube.
• Add 200 µl of sucrose.
• Add 3 µl of green dye.
• Add 3 µl of SDS detergent.
• Pipette to mix.

Exercise 3: Full Volume Transfer

• Prepare a 1.5 ml tube.
• Add 400 µl sucrose, 10 µl dye, and 10 µl SDS detergent.
• Mix.
• Transfer to a new tube containing 5 µl of dye.
• Mix.
• Note observations.

Exercise 4: Serial Dilution

• Add 30 µl of sucrose solution to each of 8 strip tubes.
• Add 30 µl of dye to the first tube.
• Perform serial transfers of 30 µl to subsequent tubes.
• Add 2 µl of dye to each tube.
• Note observations.

Exercise 5: Centrifugation of suspensions

• Prepare a 1.5 ml tube with 300 µl of suspension.
• Add 50 µl of previously prepared solution.
• Centrifuge for 5 minutes at 12000 xg.
• Remove the liquid without disturbing the sediment.
• Suspend the sediment in 150 µl of solution.

Electronic Analytical Balance

• Requires careful handling due to its precision.
• It is placed on a stable, level console to prevent shocks.

Proper Weighing Steps

• Turn on the balance.
• Tare the balance.
• Use dry, clean weighing containers.
• Close the door during weighing.
• Handle operations to avoid destabilizing the mechanism.
• Clean impurities, close the door, and turn it off after use.

Exercise 6

• Weigh a 1.5 ml tube on an analytical balance and record the measurement.
• Add water (555 uL total) to the tube and weigh again.
• Note observations.
• Attempt volume estimation using a 1000 µl pipette.

Microscopic Observations of Mitotic Divisions

• Exercise involves microscopic observations of mitotic divisions of meristematic tissue cells of Allium cepa onion using immersion.
• Students are expected to know the construction of an optical microscope and the phases of mitosis.

Optical Microscope

• A basic device in a biological laboratory combining optical and mechanical systems.
• The optical system has lighting and magnifying components.
• The mechanical system properly positions the optical elements.
• It produces a simple, magnified, and virtual image.

Optical Elements of a Microscope

• The light source was a mirror in simple microscopes but is now a built-in light bulb with a reflector; lighting should match magnification.
• The condenser consists of 2-3 lenses concentrating light onto the preparation.
• The diaphragm regulates light entering the condenser.
• Lenses magnify the image, creating a real image; distinguished as dry (up to 60x) and wet (immersion, 90-150x).
• Immersion lenses need immersion oil between the lens and preparation to increase resolution up to 0.16 µm, preventing light scattering.
• Immersion lenses are labeled "OIL" or "IMMERSION OIL".
• Ocular lenses (eyepieces) magnify the image again (2-30x), creating a virtual image; total magnification can reach 2000x.
• Resolving power is the minimum distance at which two objects can be seen separately and depends on the lens, about 0.25 µm (250 nm).

Mechanical Elements of a Microscope

• The base and arm provide structural rigidity.
• The mechanical stage secures the preparation and allows horizontal movement along the X and Y axes.
• The coarse adjustment knob adjusts the distance between the preparation and objective lens.
• It raises or lowers the stage or lenses.
• The fine adjustment is for focusing.
• The nose piece (revolver) is a rotating disk holding objective lenses for easy magnification changes.
• The head (tube) is the space between the lens and the eyepiece.
• The mechanical system adjusts the condenser's vertical position.

Cell Cycle

• The cell cycle is an ordered sequence of interphase events between mitotic divisions.
• Main phases include G1, S, G2, and M (mitosis).
• G1 and G2 are stagnation phases preparing for DNA replication or mitosis.
• DNA replication in the S phase leads to chromatid doubling.
• Functions include exact DNA doubling and precise distribution of DNA copies.
• Plays a vital role in growth, regeneration, and vegetative reproduction.

Interphase

• Interphase is the period between successive cell divisions with active metabolic changes.
• Chromosomes are non-condensed and not visible, allowing gene expression.
• Consists of G1 (pre-DNA synthesis, increased cell mass/volume), S (DNA synthesis, 2n to 4n), and G2 (pre-division, mitosis prep) phases.
• G1 and G2 phases serve as checkpoints; there is also a G0 (resting) phase where cells don't divide but retain metabolic functions.

Mitosis (M Phase)

• Mitosis is the division of a cell nucleus into two daughter nuclei in somatic cells.
• Each nucleus receives an identical set of chromosomes (diploid, 2n).
• The M phase includes karyokinesis (mitosis) and cytokinesis (cytoplasm division).

Karyokinesis Phases

• Prophase: chromatin condenses, doubled chromosomes appear, a mitotic spindle forms.
• Prometaphase: the nuclear envelope breaks down, chromosomes attach to spindle microtubules.
• Metaphase: chromosomes align on the spindle's equatorial plane.
• Anaphase: sister chromatids separate synchronously, moving to opposite poles.
• Telophase: daughter chromosomes reach spindle poles, the nuclear envelope reforms.
• Cytokinesis: the cell divides via a contractile ring of actin and myosin filaments, forming two cells, each with a nucleus.

Practical Exercises

• Use an optical microscope fluently, adjusting the image with various lenses (including immersion), documenting selected image fragments.
• Utilize theoretical knowledge of mitosis phases to find and interpret specimen images.
• Exercise 1: describe the structure of an optical microscope, indicating parts and applications.
• Exercise 2: focus an image, using a 4x lens and immersion oil, copying the selected part.
• Exercise 3: Find all mitotic division phases of the apical meristem cells of the onion Allium cepa L. root.
• Exercise 4: Draw the individual phases of mitosis for 3 pairs of chromosomes, including chromosomes with double and single chromatids occurring in the phases presented in the exercise card.
• Exercise 5: Identify the phase of cell nuclear division during or just before/after mitosis.

Learn about automatic pipettes used for measuring small liquid volumes in molecular biology labs. The exercise also covers course credit rules and lab safety. Discover accurate pipetting tips and understand the color-coded volume ranges.