Methods in Molecular Biology and Protein Chemistry: Cloning and Characterization of an Enterotoxin Subunit

Preface

Introduction and Background

Part 1 Molecular Biology

Experiment 1 Instructions for Pipetman Use

Experiment 2 Preparation of Plasmid Template DNA

Experiment 3 Estimation of Plasmid DNA Concentration
PCR Amplification of LTB Gene from Plasmid DNA

Experiment 4 Agarose Gel Eletrophoresis
Recovery of PCR Product

Experiment 5 Restriction Digest of LTB Insert
Plate Preparation

Experiment 6 Purification of Digested Insert and Vector by Agarose Gel Eletrophoresis
Recovery of Digested Insert and Vector

Experiment 7 Determination of DNA Concentrations
Ligation of Insert into Vector

Experiment 8 Transformation of Host Cells by Construct

Experiment 9 Colony-pick PCR to Check for Presence of Insert
Streak Plates with Potential Constructs
Inoculate Cultures/Run PCR

Experiment 10 Agarose Gel Electrophoresis to Verify Presence of Insert
Purification of the Plasmid Construct using a Plasmid Miniprep Kit

Experiment 11 DNA Concentration Determination
Ethanol Precipitation of Plasmid DNA
Sequencing Reaction

Experiment 12 Purification of Extension Reaction Products
Sequencing Gel Demonstration

Experiment 13 Analysis of Sequencing Data
Verification of Insert Sequence

Experiment 14 Gene Expression
Transformation into Expression Host

Experiment 15 Induction of LTB Gene Expression

Part 2 Protein Chemistry

Experiment 16 SDS-PAGE of Induction Time Course
Transfer of Protein to Membrane for Western Blot

Experiment 17 Visualization of Western Blot

Experiment 18 Large Scale Culture Preparation

Experiment 19 Isolation of LTB by Affinity Chromatography

Experiment 20 SDS-PAGE of Column Fractions
Immunoblot to verify Presence of LTB

Experiment 21 Protein Concentration
Protein Crystallization

Experiment 22 Setting up an ELISA Microtiter Plate

Experiment 23 ELISA of a Panel of Ganglioside Ligands

Experiment 24 Protein Structure Determination: X-ray Diffraction Techniques

Experiment 25 Characterization of a Protein Crystal

Experiment 26 Primer Design