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| − | ==Cell and Tissue Culture== | + | Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab.sph.umich.edu 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. |
| + | |||
| + | __NOTOC__ | ||
| + | |||
| + | ==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]] | *[[Splitting Cells]] | ||
| + | *[[Generating DMSO Stocks for Cell Culture]] | ||
*[[Differentiation of 3T3-L1 Cells]] | *[[Differentiation of 3T3-L1 Cells]] | ||
*[[Electroporation of 3T3-L1 Adipocytes]] | *[[Electroporation of 3T3-L1 Adipocytes]] | ||
*[[Fugene Transfection of 293T/COS Cells]] | *[[Fugene Transfection of 293T/COS Cells]] | ||
| − | *[[ | + | *[[Lipofectamine Mediated Knockdown]] |
| + | *[[Lipofectamine Plasmid Transfection]] | ||
*[[3T3-L1 Adipocyte Fractionation]] | *[[3T3-L1 Adipocyte Fractionation]] | ||
*[[Preparing Cell Lysates]] | *[[Preparing Cell Lysates]] | ||
*[[Glucose Uptake Assay]] | *[[Glucose Uptake Assay]] | ||
*[[Luciferase Assay]] | *[[Luciferase Assay]] | ||
| − | *[[ | + | *[[Inositol Labeling and Lipid Extraction]] |
| − | ==Cloning and Molecular Biology== | + | ===Cloning and Molecular Biology=== |
*[[Transformation of Bacteria]] | *[[Transformation of Bacteria]] | ||
*[[Mutagenesis]] | *[[Mutagenesis]] | ||
| Line 20: | Line 35: | ||
*[[Preparing an Agarose Gel]] | *[[Preparing an Agarose Gel]] | ||
*[[Cesium Chloride Preparation of DNA]] | *[[Cesium Chloride Preparation of DNA]] | ||
| + | *[[Designing and Generating CRISPR-Cas Mutants]] | ||
| − | ==Protein Analysis== | + | ===Cell Biology=== |
| + | *[[Immunofluoresence]] | ||
| + | **[[Colocalization]] | ||
| + | *[[FM4-64 Labeling of Yeast Cells]] | ||
| + | |||
| + | ===Protein Analysis=== | ||
| + | *[[Preparing a SDS-PAGE Gel]] | ||
*[[Western Blotting]] | *[[Western Blotting]] | ||
*[[Surface Plasmon Resonance - Protein Lipid Interactions]] | *[[Surface Plasmon Resonance - Protein Lipid Interactions]] | ||
| − | ===Protein Quantification=== | + | ====Protein Quantification==== |
*[[Bradford Assay]] | *[[Bradford Assay]] | ||
*[[Quantification by Absorbance at 280nm]] | *[[Quantification by Absorbance at 280nm]] | ||
| − | *[[ | + | *[[Determining Percent Purity]] |
| + | *[[Using ImageJ to Quantify Bands]] | ||
| − | ===Protein Purification=== | + | ====Protein Purification==== |
*[[French Press]] | *[[French Press]] | ||
*[[Purification of GST Fusion Proteins]] | *[[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== | + | ===Transcriptional Analysis=== |
*[[Real Time PCR From Cell Culture]] | *[[Real Time PCR From Cell Culture]] | ||
*[[Harvesting RNA from Cells grown in monolayer]] | *[[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=== | ===Genotyping=== | ||
*[[Ear Tagging and Tail Clipping]] | *[[Ear Tagging and Tail Clipping]] | ||
*[[DNA Preparation from Tail Clip]] | *[[DNA Preparation from Tail Clip]] | ||
*[[PCR Analysis of Tail DNA]] | *[[PCR Analysis of Tail DNA]] | ||
| + | *[[Colony PCR]] | ||
| + | *[[BDNF PCR]] | ||
| + | *[[TaqMan SNP Assay]] | ||
| + | |||
===Metabolic Measurements=== | ===Metabolic Measurements=== | ||
*[[Glucose Tolerance Test]] | *[[Glucose Tolerance Test]] | ||
*[[Insulin 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== | ==Figure Generation and Data Management== | ||
*[[Generation of Figures in Illustrator]] | *[[Generation of Figures in Illustrator]] | ||
| + | *[[Using Bioconductor To Analyse Microarray Data]] | ||
Latest revision as of 12:10, 15 August 2016
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
- TOPO Cloning
- Preparing an Agarose Gel
- Cesium Chloride Preparation of DNA
- Designing and Generating CRISPR-Cas Mutants
Cell Biology
Protein Analysis
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
Fly Stocks
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
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
- Perfusion of Mouse
