Hancock Laboratory Methods

Note: At the end of June, 2001, many of these methods were updated or corrected.

If you use one of these methods, please use the associated reference where listed, or an online reference format (such as that shown below which is described in the Journal of Bacteriology January 1999 Instructions to Authors):

Hancock, R. E. W. September 19 1999, posting date. [Online.]
Hancock Laboratory Methods. Department of Microbiology and Immunology,
University of British Columbia, British Columbia, Canada. http://www.cmdr.ubc.ca/bobh/methods.htm
[xx xxxxxx xxxx, date last accessed.]
 

1.  Antibiotics and Antimicrobial Peptides

Beta-lactamase induction (crude preparation)

Dinitrophenylation Assay For Polymixin B

MIC Determination by Microtitre Broth Dilution Method

Modified MIC Method for Cationic Antimicrobial Peptides

Penicillin Binding Protein Assay

 

2.  Bacterial Genetics

Alkaline Lysis Plasmid Miniprep

Blotting and Hybridization with Sodium Phosphate

Blunt End Cloning of PCR Products

Chromosomal DNA Isolation

Cosmid Library

DNA Isolation from Agarose Gels with DEAE Paper

Grid for 100 Clones

Isolation of Chromosomal DNA

Isolation of DNA from Agarose Gels with DEAE Paper

Isolation pf Phage Resistant Mutants

Lambda-TnphoA Mutagenesis of Cloned Genes in E. Coli

M13 Single-Stranded Template Preparation

Phage Resistant Mutant Isolation

Preparation of M13 Single-Stranded Template

Quick Isolation of DNA from Agarose Gels

Slot Lysis

Southern Blot

Transposon Mutagenesis of Pseudomonas Aeruginosa

Transformation of Helicobacter Pylori

Biparental Mating

CaCl₂ Transformation of E. coli

Colony Blotting with ³²P

Colony Immunoblot

CsCl-Ethidium Bromide Gradient Plasmid Preparations

 

3. Virulence Assays

 

4.  Protein Purification and Gel Electrophoresis

Acetylation (or Succinylation) of Amino Groups on Proteins

Acid-Urea Gel Electrophoresis

Crosslinking of Intact Cells

Crosslinking of Proteins

Crosslinking of Whole Cells

Electroblotting on to PVDF (Immobilon) Membrane

Estimation of Amino Groups Using TNBS

Isoelectric Focussing of Membrane Proteins by Slab Gel Method

Isolation of Outer Membrane Protein from P. aeruginosa With Octyl-POE

Isolation of Outer Membrane Protein With Zwittergent 3-14

Lipid A Preparation

Outer Membrane Protein Solubilization from P. aeruginosa with Octyl-POE

Outer Membrane Protein Solubilization with Zwittergent 3-14

Seperation of Outer and Inner Membranes

Porin F Purification

Protein and LPS Gels

Protein Assay for Detergent Solubilized Proteins

Whole Cell Protein and LPS Gels

 

5.  Liposome Methods

Liposome Preparation

Liposome Swelling Assay

Phospholipid Extraction

Planar Lipid Bilayer Experiment

Planer (Black) Lipid Bilayer Apparatus

 

6.  Outer Membranes

Cell Envelope Preparation / Solubilization

Crosslinking of Intact Cells

Crosslinking of Proteins

Crosslinking of Whole Cells

Dansyl-Polymyxin Synthesis

Dinitrophenylation Assay For Polymixin B

Inner Membrane Permeability Assay(ONPG Assay)

KDO Assay

Liposome Swelling Assay

LPS Isolation (Darveau-Hancock Method)

Methylation of Fatty Acids (Kropinski Method)

Nitrocefin Assay on Whole Cells for Outer Membrane Permeability

Nitrocefin Assay on Whole Cells for Outer Membrane Permeability Assessment

NPN Uptake Assay / Flourescence Measurements

Osmotic Shock Procedures

Outer Membrane Preparation - One Step Sucrose Gradient Procedure

Outer Membrane Protein Solubilization from P. aeruginosa with Octyl-POE

Outer Membrane Protein Solubilization with Zwittergent 3-14

Permeabilization of Whole Cells to Lysozyme

Phase Partitioning of Cells

Phosphate Uptake Assay

Planar Lipid Bilayer Experiment

Protease Digestion of Outer Membranes

Planar (Black) Lipid Bilayer Apparatus

Planar Lipid Bilayer Experiment

Synthesis of Dansyl-Polymyxin

French Press

 

7.  Biochemical Assays

Acetylation (or Succinylation) of Amino Groups on Proteins

Alkaline Phosphatase C Assay for P. aeruginosa

Alkaline Phosphatase and Phospholipase C Assays

Carbohydrate Assays

Dansyl-Polymyxin Synthesis

Dehydrogenase Assays

Dinitrophenylation Assay For Polymixin B

Estimation of Amino Groups Using TNBS

Glucosamine Rapid Assay

KDO Assay

Muramic Acid Assay

Penicillin Binding Protein Assay

Phenol Sulphuric Acid Carbohydrate Assay

Phosphate Assay

Phosphate Uptake Assay

Whole Cell Phosphate Uptake Assay

 

8.  Microbiological Techniques

Freezing Bacterial Cells

Indirect Immunofluorescence

Methionine Labelling

Oxidase Test

Phage Propagation

Media Recipes

    - BASAL MEDIUM 2

    - DEFINED PHOSPHATE MINIMAL MEDIA FOR E. coli

    - DEFINED MINIMAL MEDIA

    - DEFINED PHOSPHATE MINIMAL MEDIA (KEITH’S) - For P. aeroginosa and other bacteria

    - DULBECCO’S MINIMAL EAGLE’S MEDIA (DMEM)

    - HYBRIDOMA SELECTIVE MEDIA

    - LB (LURIA-BERTANI) MEDIUM

    - M9 MINIMAL MEDIUM

    - MINIGEL RECIPE FOR 2 GELS (1 GEL)

    - PP2 MEDIUM

Stains

    - 0.1% AMIDO BLACK

    - DESTAINING SOLUTION FOR POLYACRYLAMIDE GELS

    - LPS SILVER STAIN #1

    - LPS SILVER STAIN (CONT)

    - LPS SILVER STAIN #2

    - LPS SILVER STAIN #2 (CONT)

    - PROTEIN SILVER STAIN

    - PROTEIN SILVER STAIN

TSBD Trypticase Soy Broth Dialysate

 

9.  Host-Pathogen Interactions

In Vivo Chamber Model

Tissue Culture - Storage of Cell Lines

Tissue Culture - Thawing Cells from Liquid Nitrogen

 

10.  Functional Genomics

Transposon Mutagenesis of Pseudomonas Aeruginosa

 

11.  Immunology Based Methods

Agglutination Assays

Double Immunodiffusion

Fusion Protocol

Immunoprecipitation

Tissue Culture - Storage of Cell Lines

Tissue Culture - Thawing Cells from Liquid Nitrogen

Endotoxin Assay

Enzyme Linked Immunosorbent Assay

Source:http://www.cmdr.ubc.ca/bobh/methods.htm