Lab Practical Review Flashcards

Lab Practical Review

Lab 8: Photosynthesis

Photosynthesis Overview

  • Overall equation: 6 H2O + 6 CO2 + \text{light energy} \rightarrow C6H{12}O6 + 6 O2
  • Takes place in the chloroplast, specifically in the stroma.
  • Involves:
    • Light
    • Water
    • Carbon Dioxide
  • Key components:
    • P680
    • PQ (plastoquinone)
    • PQH2
    • Cytochrome
    • P700
    • PC (plastocyanin)
    • Oxygen-evolving complex
    • Ferredoxin (Fd)
    • Ferredoxin-NADP reductase (FNR)
    • ATP synthase
  • Light reactions convert water + photons + ADP + NADP+ to oxygen + NADPH + ATP
  • Calvin cycle uses CO2 + Water + RuBP to produce G3P + NADP+ + ADP

DPIP

  • 2,6-dichlorophenol-indophenol (DPIP) is a redox dye.
  • DPIP acts like NADP+ in the light-dependent reactions of photosynthesis.
  • It's diffusible and easily enters chloroplasts.
  • Oxidized DPIP is blue.
  • Reduced DPIP is colorless.
  • DPIP is used to study photosynthetic light reactions.

Using DPIP to study light reactions

  • Light and Chloroplast are needed for the reduction of NADP+/DPIP+.
  • In light reactions, water is used, and ATP and NADPH are produced.
  • The Calvin cycle then uses CO2 to produce sugars.
  • If DPIP replaces NADP+, DPIPH is produced instead of NADPH.

Photosynthetic Light Reactions Experiment

  • Measuring photosynthetic light reactions under different conditions.
  • Experimental conditions:
    • Fresh-Light (FL)
    • Fresh-Dark (FD) in aluminum foil
    • Boiled-Light (BL)

Photosynthetic Carbon Fixation & Respiration

  • Measuring carbon fixation and respiration.
  • A change in CO_2 indicates the rate of photosynthesis or respiration.
  • Measurements taken in the light and in the dark.
  • Consider the expected slope (+, -, or no slope) for each condition.

Lab 9: Mitosis and Meiosis

Mitosis

  • Daughter cells are genetically identical to the parental cells.
  • Know the process of mitosis: Prophase, Prometaphase, Metaphase, Anaphase, Telophase.

Meiosis

  • Daughter cells are genetically different from the parental cells.
  • Haploids result from meiosis.
  • Crossing over and independent assortment occur during meiosis.
  • Meiosis occurs in two divisions.
  • Know each phase: Prophase, Metaphase, Anaphase, Telophase for both Meiosis I and II.

Examination of Mitosis in Onion Root Cells

  • Identify each stage of the cell cycle.
  • Stages: Interphase, Prophase, Metaphase, Anaphase, Telophase.

Comparison of Mitosis and Meiosis

  • Mitosis:
    • Parent cell (2n = 6 after chromosome duplication)
    • Individual chromosomes line up.
    • Sister chromatids separate.
    • Daughter cells of mitosis are 2n.
  • Meiosis:
    • Parent cell (2n = 6 after chromosome duplication)
    • Pairs of homologous chromosomes line up (Chiasma formation in Prophase I).
    • Homologs separate in Meiosis I.
    • Daughter cells of meiosis I are n.
    • Sister chromatids separate in Meiosis II.
    • Daughter cells of meiosis II are n.

Lab 10: Monohybrid and Dihybrid Crosses

Monohybrid Cross (Mendel's experiment)

  • P generation: TT (tall) x tt (short).
  • F1 generation: All Tt (tall).
  • F2 generation: Phenotypic ratio of 3 tall : 1 short.
  • Punnett square is used to determine offspring genotypes.
  • Test cross: Cross an unknown plant with a homozygous recessive (tt) to determine if it's homozygous dominant (TT) or heterozygous (Tt).

Dihybrid Cross

  • Cross: YyRr x YyRr (Y = yellow, y = green, R = round, r = wrinkled).
  • Punnett square is used to determine offspring genotypes.
  • Phenotypic ratio: 9 Yellow-round : 3 Green-round : 3 Yellow-wrinkled : 1 Green-wrinkled (Assuming Complete Dominance).

Important Concepts

  • Chromosomal Theory of Inheritance: Independent Assortment & Law of Segregation.
  • Dominant and Recessive Alleles.
  • Phenotype vs. Genotype.
  • Phenotypic vs. Genotypic Ratios.
  • Karyotype.
  • Homozygous Dominant vs. Heterozygous.
  • Test Cross.
  • Monohybrid vs. Dihybrid Crosses.
  • P, F1, and F2 Generations.
  • Pedigree.
  • Autosomes and Sex Chromosomes.
  • Linked Genes and Sex Linkage.

Lab 11: Restriction Digestion and Gel Electrophoresis

Restriction Enzymes

  • Restriction enzymes cut DNA at specific sites.
  • Examples: BsmB I, Eco0109 I, Aat II, SspI, Eco57 I, Xmn I, Bcg I, Sca I, Drd I, PvuI, Ava II, BsrD I, Fsp I, BglI, Bpm I, BsrF1, Bsa I, Nde I, Nar I/Kas I, Bgl I, Fsp I, Pvu II
  • pUC19 plasmid: 2686 bp with polylinker cloning sites (396-454).

Gel Electrophoresis

  • Visualizing DNA fragments.
  • Example calculation: 2686 bp - 322 bp = 2364 bp.
  • Example: Fragments with sizes 2464, 2364, 1209, 933, 322, 222 and 81 base pairs.
  • Example: Lambda fragments with sizes 23130 bp, 9416 bp, 6557 bp, 4361 bp, 2322 bp, 2027 bp and 564 bp.
  • Gel 1 description: 1% agarose stained in Ethidium Bromide, 0.5 µg/lane, 1X TAE.
  • Gel 2 description: DNA ladder with bands at 22882, 4000, 3000, 2000, 1500, 1000, 850, 650, 500, 400, 300, 200, and 100 bp.

Lab 12: lacZ Gene and Gene Expression

lac Operon

  • Normal Conditions (no lactose): "off state".
    • Active repressor binds to the operator, preventing RNA polymerase from transcribing lacZ.
  • In the Presence of Lactose: "on state".
    • Lactose binds to the repressor, inactivating it and allowing RNA polymerase to transcribe lacZ.
  • In the Presence of IPTG: "on state".
    • IPTG (Isopropylthiogalactoside) is a lactose analog that inactivates the repressor, allowing transcription of lacZ.
    • IPTG acts as lactose in experiment.

Experiment Results

  • Measure Beta-galactosidase activity to determine gene expression levels in different tubes and controls.

General Lab Knowledge

Review

  • Lab safety concepts.
  • Lab 8: Experimental treatments, observations, and their meanings. Photosynthesis and respiration equation.
  • Lab 9: Differences between all mitosis and meiosis phases, outcomes, genetic identity. Function of crossing over and independent assortment.
  • Lab 10: Monohybrid and dihybrid crosses, phenotypic and genotypic ratios, gene linkage.
  • Lab 11: Steps in the experiment, DNA location during each step. Results of cutting linear or circular DNA, and interpreting gel fragment sizes.
  • Lab 12: lacZ gene, transcription and translation, role of IPTG and ONPG, interpreting experiment results for gene expression.