Main Page

Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the Bridges Lab Website. All of our experimental protocols are available through this site and are free to use/copy/modify by anyone without any restrictions. You can locate protocols by looking at this index, searching by category (see below) or searching by keyword. If you notice an error, would like to discuss these methods or would like to be able to edit these pages contact us via email.

Protocol Categories

See Special:Categories for as listing of protocols by category.

Lab Safety

• For notes about required training courses see Safety and Animal Training
• For lab-specific standard operating procedures see Category: SOP

Common Protocols

Cell and Tissue Culture

• Splitting Cells
• Generating DMSO Stocks for Cell Culture
• Differentiation of 3T3-L1 Cells
• Electroporation of 3T3-L1 Adipocytes
• Fugene Transfection of 293T/COS Cells
• Lipofectamine Mediated Knockdown
• Lipofectamine Plasmid Transfection
• 3T3-L1 Adipocyte Fractionation
• Preparing Cell Lysates
• Glucose Uptake Assay
• Luciferase Assay
• Inositol Labeling and Lipid Extraction

Cloning and Molecular Biology

• Transformation of Bacteria
• Mutagenesis
• PCR Amplification of DNA
• Restriction Enzyme Based Cloning
  • Restriction Enzyme Based Cloning - Ordering Primers
• TOPO Cloning
• Preparing an Agarose Gel
• Cesium Chloride Preparation of DNA
• Designing and Generating CRISPR-Cas Mutants

Cell Biology

• Immunofluoresence
  • Colocalization
• FM4-64 Labeling of Yeast Cells

Protein Analysis

• Preparing a SDS-PAGE Gel
• Western Blotting
• Surface Plasmon Resonance - Protein Lipid Interactions

Protein Quantification

• Bradford Assay
• Quantification by Absorbance at 280nm
• Determining Percent Purity
• Using ImageJ to Quantify Bands

Protein Purification

• French Press
• Purification of GST Fusion Proteins
• Affinity Purificatoin of Antibodies (Coupling to Activated CH Sepharose 4B)
• Preparation of DIG Labelled Probes
• Glycogen Synthase Assay
• Triglyceride Assay from Cells and Tissues
• Triglyceride Assay from Drosophila (German Method)

Transcriptional Analysis

• Real Time PCR From Cell Culture
• Harvesting RNA from Cells grown in monolayer
• Using Bioconductor To Analyse Beadarray Data
• Using Bioconductor To Analyse Microarray Data

Mouse/Fly Protocols

• Maternal Particulate Exposure Breeding

Fly Stocks

• Maintenance of 18C Fly Stocks
• Fly Food Protocol

Genotyping

• Ear Tagging and Tail Clipping
• DNA Preparation from Tail Clip
• PCR Analysis of Tail DNA
• Colony PCR
• BDNF PCR
• TaqMan SNP Assay

Metabolic Measurements

• Glucose Tolerance Test
• Insulin Tolerance Test
• Measuring Fasting Insulin
• Monitoring Food Intake

Tissue Preparation

• Harvesting Mouse Tissue
• Preparation of Protein Lysates from Mouse Tissues
• Preparation of RNA Samples from Mouse Tissues
• Paraffin Embedding of Tissue Samples
• H&E Staining of Mouse Tissue
  • Adipocyte Cell Counting with ImageJ
• Perfusion of Mouse

Media and Buffers

Yeast Media

• YPD Media and Agar
• Yeast Selective Media and Agar

Buffers

Lysis Buffers

• KRBH Buffer
• RIPA Buffer
• CHAPS Lysis Buffer

SDS-PAGE Buffers

• SDS-PAGE Separating Gel Buffer
• SDS-PAGE Stacking Gel Buffer
• TORC1 Kinase Buffer
• Acrylamide Solution

Other Buffers

• KRBH Buffer
• MTORC1 Kinase Assay
• TORC1 Kinase Buffer

Figure Generation and Data Management

• Generation of Figures in Illustrator
• Using Bioconductor To Analyse Microarray Data