Main Page

Visit our Lab's Webpage at www.phagocytes.ca

Welcome to the protocol wiki for the lab of Dr. Bryan Heit, at the University of Western Ontario. This site contains the protocols, and other information, we frequently use in our lab.  Only laboratory members may edit pages, but anyone is welcome to use these protocols.  If you use these protocol, please cite us.  A link for generating citations can be found on the left side of the page.

Consult the User's Guide for information on using the wiki software.

Lab members:

• To get a wiki account please contact Dr. Heit.
• Log in (upper-right side of screen) to add/edit pages.  
• Please follow these formatting instructions when editing/creating protocols.
• To create a new page, follow these instructions.

Index of Protocols

Lab Operations Entrance Protocol Exit Protocol Guidelines for Proper Use of Laboratory Notebooks Proper Saving of Experimental Files Setting up Access to Network Drives & Wiki	DNA/Cloning Colony PCR PCR Digests Ligation Gibson Assembly Quick 'n' Easy Competent E. coli (Dh5a) Generating Competent E. coli (TFB, BL21 & ZYCY10P3S2T) Transducing E. coli Transformation Neon® Transfection System Generating Minicircles	Microscopy Competition of Charged Molecules with Lipophilic Cations Fluorescent Immunostaining Inhibition of Focal Contact Signaling Single Particle Tracking Staining for GSD Reducing Photobleaching Acid Washing Coverslips Live Cell FRET 3D Printed PDMS Chambers
General Protocols Common Buffers Cell Culture Guidelines Bacterial Growth Media Primary Macrophage Culture Freezing and Thawing Cells Raw Cell Transfection J774 Cell Transfection Antibiotics Antibiotic Plates Agarose Gels Water Bath Antibiotic Solution	Protein work Western Blotting Immunoprecipitation Fab preparation Fab purification using FPLC Updated FPLC Size Exclusion Procedure Coomassie Staining Receptor activation by Cross-Linking Stripping & Reprobing Blots	Lipids Asymmetric liposomes Lipid Coated Bead Preparation Lipid Extraction from Cells Lipisome and Lipid-Coated Beads
Phagocytosis Protocols Opsonization Preparation of Silica-Magnetic Beads Synchronised Phagocytosis Phagosome Isolation Primary Human Macrophages Labelled E coli Inside-out Labelling of Bacteria Gentamicin Protection Assay Preparation of Digestion-Tracking Bacteria	Cell Biology Ablation of Recycling Endosomes Cell-Type Specific Transfection Protocols G418 & Puromycin Kill Curves Apoptosis Detection with AnnexinV and PI

Lab Operations Entrance Protocol Exit Protocol Guidelines for Proper Use of Laboratory Notebooks Proper Saving of Experimental Files Setting up Access to Network Drives & Wiki General Protocols Common Buffers Cell Culture Guidelines Bacterial Growth Media Primary Macrophage Culture Freezing and Thawing Cells Raw Cell Transfection J774 Cell Transfection Antibiotics Antibiotic Plates Agarose Gels Water Bath Antibiotic Solution Phagocytosis Protocols Opsonization Preparation of Silica-Magnetic Beads Synchronised Phagocytosis Phagosome Isolation Primary Human Macrophages Labelled E coli Inside-out Labelling of Bacteria Gentamicin Protection Assay Preparation of Digestion-Tracking Bacteria

DNA/Cloning Colony PCR PCR Digests Ligation Gibson Assembly Quick 'n' Easy Competent E. coli (Dh5a) Generating Competent E. coli (TFB, BL21 & ZYCY10P3S2T) Transducing E. coli Transformation Neon® Transfection System Generating Minicircles Protein work Western Blotting Immunoprecipitation Fab preparation Fab purification using FPLC Updated FPLC Size Exclusion Procedure Coomassie Staining Receptor activation by Cross-Linking Stripping & Reprobing Blots Cell Biology Ablation of Recycling Endosomes Cell-Type Specific Transfection Protocols G418 & Puromycin Kill Curves Apoptosis Detection with AnnexinV and PI

Microscopy Competition of Charged Molecules with Lipophilic Cations Fluorescent Immunostaining Inhibition of Focal Contact Signaling Single Particle Tracking Staining for GSD Reducing Photobleaching Acid Washing Coverslips Live Cell FRET 3D Printed PDMS Chambers Lipids Asymmetric liposomes Lipid Coated Bead Preparation Lipid Extraction from Cells Lipisome and Lipid-Coated Beads

---

Links to More Protocols

General Protocol Sites BenchFly - Free Video Protocols Protocols Online - large database of biology protocols Open Wet Ware - large database of open protocols Molecular Station - links to many lab-generated protocols TheLabRat - various protocols & lab resourses. Common Buffer Recipes @ TheLabRat Chips & Tips - Microfluidics protocols

Free Science Ebooks Analyzing fluorescence microscopy images with ImageJ Bioimage Data Analysis - Free registration required

Useful Links

Molecular Biology Databases Pubmed Gene - Find gene information Pubmed RefSeq - Find reference sequences Uniprot - Find protein sequence & structure HapMap - Find human polymophisms OMIM - Find human gene-assocations PANTHER - Automated Protein Function Classification BioGrid - Protein Interactions Ensemble Genome - Browse multiple genomes Human Protein Atlas - Info onn gene exrpession, antibody's, etc Gene Cards - Condenced information on genes iHOP - Information Hyperlinked Over Proteins Human Protein Reference Database PDB - Protein Structures PolyPhen2 - SNP Phenotype Predictor SIFT - SNP Phenotype Predictor/db TimeTree - evolutionary divergence database Journal Resources Pubmed Pubmed Central (USA) Pubmed Central (Canada) Google Scholar Eigenfactor - free journal imapct scores Genomics Resources GWAS Central

Molecular Biology Tools NCBI Blast ExPASy Tools OligoCalc - PCR primer Tm calculator NEB Cutter NEB Isoschizomers Reverse-Complement DNA Sequence Ligation Calculator AddGene - clone by e-mail! PattenProt - search genomes for protein patterns Biology Workbench EMBL Sequence Tools NCBI ORF Finder BCM Search Launcher SWISS Model protein modeling Secondary Structure Prediction PredictProtein - Protein structure prediction STRAP - Protein aligments with structure In Vivo Tools EMAP - Virtual mouse anatomy Mouse phenome database - mouse phenotypes

Microscopy Tools Microscopy U - Everything you want to know about microscopes Paper on Image Processing Standards - How not to loose your job Fluorophore Spectra Viewer at Life Technology Fluorescent Spectra Database - FRET and other Yet More Spectra - from Arizona University www.confocal-microscopy.org - Data on LSM methods and equipment FIJI - FREE imageJ based image processing program Free image processing textbook Lectures on image processing VAA3D FREE viewer for large 3/4/5D datasets Protease Tools MEROPS - Peptidase database PMAP - Proteolysis Map CASVM - Caspace substrate prediction CASBAH - Caspase cleaveage site database Lipid Tools Lipid Maps - Lipidomics gateway at Nature Avanti Lipids - Buy lipids Chemical Tools ChemSpider - General chemistry database PubChem - Chemical structure database