bio lab

1. Microscope Use

  • Starting:

    • Start on lowest objective lens (typically 4x).

    • Use coarse adjustment to bring the stage up until you see the specimen.

    • Switch to fine adjustment to sharpen focus.

  • Finishing:

    • Lower the stage.

    • Switch back to lowest power.

    • Turn off light, wrap cord neatly, cover microscope.

  • Functions of parts:

    • Ocular lens: Eyepiece (usually 10x)

    • Objective lens: Primary magnification

    • Stage: Where slide is placed

    • Coarse knob: Large adjustments

    • Fine knob: Precise focusing

    • Diaphragm: Adjusts light

  • Finding and Focusing on Tissue:

    • Always begin at low power and center the tissue before switching to a higher magnification.

    • Higher magnification → smaller field of view and less depth of focus.

  • Identifying Stages of Mitosis:

    • Interphase = normal nucleus

    • Prophase = chromosomes visible

    • Metaphase = chromosomes lined at middle

    • Anaphase = chromosomes pulled apart

    • Telophase = new nuclei forming

2. Solution Making and Measurements

  • Units to Know:

    • Molarity (M) = moles solute / liter solution

    • Percent Solutions (%) = (grams solute / mL solvent) × 100

    • Dilutions: Use C₁V₁ = C₂V₂ to solve.

  • Measurement Tools:

    • Micropipette: volumes < 1 mL (μL)

    • Serological pipette: 1–10 mL

    • Graduated cylinder: 10–1000 mL

    • Beaker: rough measurements, not highly accurate

  • Micropipette Rules:

    • Always use correct size (P10, P100, P1000).

    • Use new sterile tips.

    • Press plunger to first stop before entering liquid, release slowly.

3. Spectrophotometer Use

  • Purpose: Measures absorbance of light by a solution.

  • Calibrating:

    • Use a blank (often water or buffer) to zero the machine before measuring.

  • Wavelength:

    • Set based on absorption spectrum; λmax = wavelength where maximum absorption occurs.

  • Absorbance vs. Concentration:

    • Higher absorbance = Higher concentration (direct relationship).

  • Errors:

    • Dirty cuvette = bad readings

    • Incorrect blank = incorrect zero = all future measurements wrong

🧪 LAB TOOLS You Must Know:

Tool

Function

Centrifuge

Separates by density (heavy parts to bottom)

Vortex mixer

Mixes solutions

Beaker

Holding/mixing liquids (rough measurement)

Graduated Cylinder

Accurate liquid measurement

Serological Pipette

Transfer measured volumes (1–10 mL)

Micropipette

Transfer small volumes (μL range)

Microcentrifuge Tube

Holds small samples during centrifugation

Gel Comb/Mold

Makes wells in gel for electrophoresis

Electrophoresis Chamber

Runs electric current through gel

Spectrophotometer

Measures light absorbance

Cuvette

Holds samples in spectrophotometer

🧫 Solutions You Used and What They Do:

Solution

Function

dH₂O

Solvent, calibration

CTAB

Breaks open cells (DNA extraction)

Chloroform

Separates organic layer (DNA extraction)

Isopropanol/Ethanol

Precipitates DNA

Tris-HCl

Buffer for stable pH

Microbeads

Break cell walls (mechanical lysis)

Agarose

Makes gel for electrophoresis

TAE buffer

Conducts electricity during electrophoresis

Gel Red

Stains DNA so it's visible under UV

ONPG

Substrate for lactase enzyme

ONP

Yellow product measured

Na₂CO₃

Stops enzyme reaction

PBS

Buffer for enzymatic reactions

📊 DATA ANALYSIS, STATISTICS, AND GRAPHING:

1. Summary vs Raw Data

  • Raw Data = Individual experimental results.

  • Summary Data = Mean, SD across replicates.

2. Statistical Terms

  • Mean = average

  • Standard deviation (SD) = variability of data points around mean

3. Graphing

  • Scatter plot = continuous data

  • Column graph = categorical data

  • Always include:

    • X and Y axes labels (with units!)

    • Group labels

    • Error bars (± SD)

    • Figure legend (explains graph)

🔬 EXPERIMENTAL DESIGN:

Concept

What to Know

Research Question

The problem you're investigating

Hypothesis

Tentative answer to the question

Prediction

"If [hypothesis], then [expected result]"

Independent Variable

What you change

Dependent Variable

What you measure

Controlled Variables

What stays the same

Sample Size

Number of subjects/samples in a group

Replicates

Repeated trials to reduce error

🧬 SPECIFIC LABS TO KNOW:

1. DNA Extraction:

  • Steps:

    1. Break cell membranes (CTAB + vortex + beads).

    2. Separate layers (centrifuge + chloroform).

    3. Precipitate DNA (isopropanol).

    4. Wash DNA (ethanol).

  • Layer Separation:

    • Aqueous layer = DNA

    • Organic layer = proteins, lipids

  • Key Concepts:

    • Alcohol precipitates DNA because DNA is insoluble in alcohol.

2. Gel Electrophoresis:

  • Movement:

    • DNA moves toward positive electrode (DNA = negatively charged).

    • Smaller fragments move faster through gel.

  • Key Substances:

    • Agarose: Gel material

    • TAE buffer: Conducts electricity

    • Gel Red: Stains DNA for visibility

  • Band Interpretation:

    • Bands = DNA fragments

    • DNA ladder = size standard

    • More bands = more cut sites

3. Enzyme Reaction (ONPG Assay):

  • Reaction:

    • Lactase enzyme breaks ONPG into ONP (yellow).

  • Measurement:

    • Measure ONP concentration by absorbance at 420 nm.

  • Purpose of Chemicals:

    • PBS = buffer

    • Na₂CO₃ = stops reaction

  • Rate Calculation:

    • Amount of ONP formed / time

4. Mitosis (Onion Root Tip):

  • Root Tip Zones:

    • Meristem = where most division occurs

  • Stages of Cell Cycle:

    • Interphase = Growth and DNA replication

    • Mitosis = Nuclear division (prophase, metaphase, anaphase, telophase)

  • Analysis:

    • Count # of cells in each stage to estimate time spent per phase.