Biol 1406 Lab Review_2(1)(1) - Tagged

Lab Exam Review Overview

  • Exam covers Lab 7 to Lab 9.

Lab 7: Thin Layer Chromatography (TLC)

  • Stationary Phase: Silica Plate

    • Silica is charged and polar.

  • Mobile Phase: Ether and Acetone

    • Travels along the Silica plate, carrying pigments.

  • Test Sample: Spinach extract applied to the plate for pigment separation.

  • Function of Pigments: Harvest energy from light to convert into chemical energy (food).

  • Absorption Spectrum: Definition needed to understand light absorption by pigments.

Results of TLC

  • Pigments Identified:

    • Carotene

    • Pheophytin

    • Chlorophyll a

    • Chlorophyll b

    • Light

    • Xanthophyll

  • Result source: Google.com.

General Procedure of TLC

  • Specific procedural details can be elaborated upon but involve applying extracts to a silica plate and using solvent to separate components based on polarity.

Absorption Maxima

  • Absorption Spectrum: Measures relative light absorption across wavelengths.

    • Chlorophyll a & b: Absorb light at varying wavelengths, resulting in multiple peaks in spectrum.

    • Absorption Maximum: Individual peaks indicating specific wavelengths of maximum absorption.

Beer-Lambert Law

  • Linear Relationship: Between absorbance and concentration of an absorbing species.

  • Equation:

    • Absorbance (A) = e * L * c

    • e = molar extinction coefficient,

    • L = path length of cell holder,

    • c = concentration of solution.

Lab 8: Column Chromatography

  • Method: Size exclusion chromatography using Sephadex column to purify GFP and BFP.

  • Quantification Method: Bradford protein assay to determine concentrations of purified proteins.

Bradford Assay Steps

  • Standard Curve: Created with known protein concentrations.

  • Reagent Use: Bradford Reagent reacts with proteins; the product is blue.

  • Standard Protein: Bovine Serum Albumin (BSA), initially colorless.

  • Color Interpretation: Higher protein concentration equals more blue product measured at 595 nm absorbance.

Standard Curve Equation for Protein Concentration

  • Equation: y = mx + b

    • y = Absorbance at 595 nm

    • m = slope of the line

    • x = protein concentration

    • b = y-intercept.

Green Fluorescence Protein (GFP) Information

  • Source: Isolated from Aequorea victoria.

  • Characteristics:

    • 239 amino acids,

    • Absorbs at 395 nm,

    • Molecular weight: 26,290 Daltons.

  • Chromophore Formation: Involves amino acids Ser, Tyr, Gly undergoing cyclization.

  • Mutations: 3-point mutations enhance hydrophilicity improving performance.

  • Excitation Source: UV-light beneficial for chromophore excitation.

SDS-PAGE Overview

  • Sieve Function: Polyacrylamide gel acts as a sieve for protein separation.

  • Current Application: Electrical current applied, utilizing Laemmli Buffer for protein stabilization.

  • Key Components:

    • SDS: detergent aiding separation,

    • Glycerol: increases density for proper migration,

    • DTT: reduces disulfide bridges, linearizing proteins,

    • Bromophenol Blue: dye to track sample migration.

Gel Electrophoresis Basics

  • Process: Separates molecules by size and charge using gel medium.

  • Application: DNA and protein samples analyzed under electric current.

  • Buffers: Balance charges helping migration.

Role of SDS and Heat in Denaturation

  • SDS Function: Provides a negative charge to proteins, maintaining solubility and denaturing proteins.

  • Heat Application: Further denatures proteins, affecting migration size.

Molecular Weight Determination via Standard Curve

  • Protein Marker: Use of Bio-Rad standards for plotting standard curve

  • Graphing:

    • X-Axis: Distance migrated (mm)

    • Y-Axis: Log(Molecular Weight).

Lab 9: Genetically Modified Organisms (GMOs)

  • Examples of GMOs:

    • Summer Squash, Tomato, Rice, Sweet Corn, Canola Oil, Yeast, and more.

    • Insights on current market status and agricultural implications discussed.

  • Contamination Concerns: GMO products contaminating non-GMO crops significantly.

Genetic Modification Process Steps

  • Steps Overview:

    1. Identify and isolate desired genetic trait.

    2. Insert trait into new genome using various methods including electroporation and gene guns.

  • Promoters and Terminators:

    • Common promoter: 35S from Cauliflower Mosaic Virus; uses specific sequence lengths for function.

Polymerase Chain Reaction (PCR)

  • Essential Chemicals:

    • Primers, DNTPs, DNA Taq Polymerase, DNA template required for reactions.

  • Reactions Context: Carried out to test for GMO presence in food samples.

Gel Electrophoresis Application in PCR

  • Types of Gels:

    • Protein Gel for proteins

    • Agarose Gel for DNA.

  • Separation Mechanism: Based on size and charge under electrical current.

Sample Questions for Preparation

  • Tools used for separating pigments?

Column Chromatography, a technique that utilizes a stationary phase and a mobile phase to separate components based on their interactions with each phase.

  • Basic principle of PCR?

    The Basic Principles of a PCR is that it involves the amplification of specific DNA sequences through repeated cycles of denaturation, annealing, and extension, allowing for exponential increases in the target DNA.

  • Gel Electrophoresis

    a method that separates nucleic acids or proteins based on their size and charge, allowing for visualization and analysis of the separated components.

  • Spectrophotometry:

    A technique used to measure the amount of light absorbed by a sample, providing quantitative data about the concentration of nucleic acids or proteins.

  • Real-Time PCR:

    An advanced method that allows for the monitoring of the PCR process in real-time, providing quantitative data as the reaction progresses.

  • Cloning:

    A molecular technique used to create copies of specific DNA fragments, which can be used for various applications such as gene expression studies, protein production, or genetic modification.

  • DNA Sequencing:

    A process that determines the precise order of nucleotides within a DNA molecule, enabling researchers to analyze genetic information and identify mutations.

  • Working principle of Column Chromatography?

    The working principle of Column Chromatography are based on the differential adsorption of compounds to the stationary phase while being carried through the column by a mobile phase, allowing for the separation of mixtures based on their affinities for each phase.

  • Differences between GFP and BFP?

    The main differences between GFP and BFP are their fluorescence properties and the specific wavelengths of light they emit. GFP (Green Fluorescent Protein) emits green light when excited by ultraviolet or blue light, while BFP (Blue Fluorescent Protein) emits blue light under similar conditions. Additionally, GFP is often used as a marker in molecular and cellular biology due to its brightness and stability, whereas BFP is less commonly used but can be useful for applications requiring blue fluorescence.

  • Mechanism of Electrophoresis?

    The mechanisms of column chromatography involve the separation of compounds based on their affinity for the stationary phase versus the mobile phase, allowing for the isolation of specific components from a mixture.

  • Methods for measuring DNA or Protein fragments on the gel?

    The methods for measuring DNA or Protein Fragments on a gel include the use of spectrophotometry, fluorometry, and densitometry, each providing quantitative analysis of the bands formed after electrophoresis.

  • Strategies to test food for GMO content?

    Strategies include polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and next-generation sequencing (NGS), which allow for the detection of specific genetic markers associated with genetically modified organisms.

  • Controls utilized in the GMO lab?

    Controls utilized in the GMO lab involve the use of both positive and negative controls to ensure the accuracy of test results. Positive controls contain known GMO content to validate the detection methods, while negative controls lack GMO content to confirm that any detected signals are not false positives.

  • How to interpret gel results accurately?

    To interpret gel results accurately, one must compare the bands observed on the gel to a molecular weight marker, ensuring that the size of the bands corresponds to the expected sizes of the DNA fragments associated with the target genetic markers. Additionally, the intensity of the bands can provide insight into the relative abundance of the DNA in the sample.