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| − | Welcome to the Bridges Lab protocol site. If you would like to know more about our group, please visit the [http://bridgeslab. | + | 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__ | __NOTOC__ | ||
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See [[Special:Categories]] for as listing of protocols by category. | See [[Special:Categories]] for as listing of protocols by category. | ||
| − | ==Cell and Tissue Culture== | + | ==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]] | *[[Generating DMSO Stocks for Cell Culture]] | ||
| Line 20: | Line 26: | ||
*[[Inositol Labeling and Lipid Extraction]] | *[[Inositol Labeling and Lipid Extraction]] | ||
| − | ==Cloning and Molecular Biology== | + | ===Cloning and Molecular Biology=== |
*[[Transformation of Bacteria]] | *[[Transformation of Bacteria]] | ||
*[[Mutagenesis]] | *[[Mutagenesis]] | ||
| Line 29: | 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]] | ||
| − | ==Cell Biology== | + | ===Cell Biology=== |
*[[Immunofluoresence]] | *[[Immunofluoresence]] | ||
**[[Colocalization]] | **[[Colocalization]] | ||
*[[FM4-64 Labeling of Yeast Cells]] | *[[FM4-64 Labeling of Yeast Cells]] | ||
| − | ==Protein Analysis== | + | ===Protein Analysis=== |
*[[Preparing a SDS-PAGE Gel]] | *[[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]] | ||
| Line 45: | Line 52: | ||
*[[Using ImageJ to Quantify Bands]] | *[[Using ImageJ to Quantify Bands]] | ||
| − | ===Protein Purification=== | + | ====Protein Purification==== |
*[[French Press]] | *[[French Press]] | ||
*[[Purification of GST Fusion Proteins]] | *[[Purification of GST Fusion Proteins]] | ||
| Line 54: | Line 61: | ||
*[[Triglyceride Assay from Drosophila (German Method)]] | *[[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]] | ||
| Line 60: | Line 67: | ||
*[[Using Bioconductor To Analyse Microarray Data]] | *[[Using Bioconductor To Analyse Microarray Data]] | ||
| − | ==Mouse/Fly Protocols== | + | ===Mouse/Fly Protocols=== |
| − | ==Fly Stocks== | + | *[[Maternal Particulate Exposure Breeding]] |
| + | |||
| + | ===Fly Stocks=== | ||
*[[Maintenance of 18C Fly Stocks]] | *[[Maintenance of 18C Fly Stocks]] | ||
*[[Fly Food Protocol]] | *[[Fly Food Protocol]] | ||
| Line 70: | Line 79: | ||
*[[PCR Analysis of Tail DNA]] | *[[PCR Analysis of Tail DNA]] | ||
*[[Colony PCR]] | *[[Colony PCR]] | ||
| + | *[[BDNF PCR]] | ||
| + | *[[TaqMan SNP Assay]] | ||
===Metabolic Measurements=== | ===Metabolic Measurements=== | ||
| Line 77: | Line 88: | ||
*[[Monitoring Food Intake]] | *[[Monitoring Food Intake]] | ||
| − | ==Tissue Preparation== | + | ===Tissue Preparation=== |
*[[Harvesting Mouse Tissue]] | *[[Harvesting Mouse Tissue]] | ||
*[[Preparation of Protein Lysates from Mouse Tissues]] | *[[Preparation of Protein Lysates from Mouse Tissues]] | ||
*[[Preparation of RNA Samples 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== | + | ===Media and Buffers=== |
| − | ===Yeast Media=== | + | ====Yeast Media==== |
*[[YPD Media and Agar]] | *[[YPD Media and Agar]] | ||
*[[Yeast Selective Media and Agar]] | *[[Yeast Selective Media and Agar]] | ||
| − | ===Buffers=== | + | ====Buffers==== |
| − | ====Lysis Buffers==== | + | =====Lysis Buffers===== |
*[[KRBH Buffer]] | *[[KRBH Buffer]] | ||
*[[RIPA Buffer]] | *[[RIPA Buffer]] | ||
*[[CHAPS Lysis Buffer]] | *[[CHAPS Lysis Buffer]] | ||
| − | ====SDS-PAGE Buffers==== | + | =====SDS-PAGE Buffers===== |
*[[SDS-PAGE Separating Gel Buffer]] | *[[SDS-PAGE Separating Gel Buffer]] | ||
*[[SDS-PAGE Stacking Gel Buffer]] | *[[SDS-PAGE Stacking Gel Buffer]] | ||
*[[TORC1 Kinase Buffer]] | *[[TORC1 Kinase Buffer]] | ||
*[[Acrylamide Solution]] | *[[Acrylamide Solution]] | ||
| − | ====Other Buffers==== | + | =====Other Buffers===== |
*[[KRBH Buffer]] | *[[KRBH Buffer]] | ||
*[[MTORC1 Kinase Assay]] | *[[MTORC1 Kinase Assay]] | ||
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
