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Lab Manual

Please feel free to try any of our protocols.  They are available in web and Adobe Acrobat format.  All that we ask that you credit us (or the original author) when using them.  This document is in a permanent state of flux - no protocol is ever really perfect and therefore it is never finished!


BIOLOGICAL REAGENTS

ER / MITO SEPARATION

Separation of Mitochondria and Microsomes

Preparation of Heavy and Light Membrane Fractions From MDCK Cells


ASCITES FLUID

Collecting Ascitic Fluid


ANTIBODIES IN RABBITS

Summary of Rabbit Immunization Procedures

Affinity Purifying Antibody from Rabbit Serum

Purifying Antibodies Made using a GST-fusion Protein


CANINE MICROSOMES

Procedure for Preparing Microsomal Membranes from Dog Pancreas


IgY FROM EGG YOLK

Preparation of IgY from chicken egg yolk


CLL CELL

Ficoll gradient procedure for CLL-cells

P100 membrane prep. of CLL-cells


RIBOSOME PREP

Ribosome(R), S-100 enzymes(S), and peptidyl-tRNA hydrolase(H) preps


CELL FREE SYSTEMS

TRANSCRIPTION

SP6 Cell Free Transcription

Set Up Reaction

Order of Setup and Incubation

Reagents

Buffers

Sample Protocol

Transcription Reactions

Troubleshooting

Transcription Kinetics


TRANSLATION

General Cell Free Translation

Reticulocyte Lysate and Wheat Germ Extracts

Rabbit Reticulocyte Lysate Cell-Free Translation

Nuclease Treatment and Desalting

Translation Pausing

Ribosome Pausing Assay

RPFs from Free and Membrane Bound Ribosomes

tRNA Crosslinking

Preparative Aminoacylation of Pure tRNA

Phenol Extraction of tRNA

Generation of Integration Intermediates

Chemical Modification of Lys-tRNA

Immunoprecipitation of translation products

Translation in RRL

Buffers

Sample Protocol

Troubleshooting

Pause Assay

RPFs from Free and Membrane Bound Ribosomes

History and evolution of cell-free protein synthesizing systems

Import Into Mitochondria

Wheat Germ Cell-Free Translation

Protocol for wheat germ cell-free translation

Wheat Germ Extract Buffers and reagents

Sample WG Protocol

Wheat Germ Extract Recipes

Troubleshooting Wheat Germ Translation Reactions

Preparation of a wheat germ extract

E coli Translation

Translation Protocol

Sample Protocol

Troubleshooting

Lysate Preparation of S-30

Membrane-Free Ribosomes (mfrs)

Ribosomal Pellet

Dialysis Tubing Work-up

Membrane-Free Translation System

Buffers and Reagents


TARGETING

Targeting and Translocation

Proteolysis Assays for Translocation

Post Translational Targeting to RMs

Endoglycosidase H

Carbonate Extraction

Separating Membrane-Targeted Molecules using Gel Filtration

Pelleting Assays


DNA

DNA ISOLATION AND PLASMID PURIFICATION

Phenol extraction and ethanol precipitation.

Large Scale Plasmid Prep for 100 ML Superbroth Cultures

Plasmid Miniscreen Protocol (Boil Prep)

Alkaline Lysis Miniprep

Alkaline Lysis Miniprep Modified 1

Alkaline Lysis Miniprep Modified 2

Amplification of plasmids with chloramphenicol

Single-Strand DNA Prep. Large Scale

Titre of Bacteriophage

Large Scale Bacteriophage Prep


LIGATION


RESTRICTION DIGESTS

Restriction Endonuclease Digestion

Large Scale Digest Prep for Construction


AGAROSE GEL ELECTROPHORESIS

ISOLATION OF DNA FRAGMENTS

Elution of DNA from Agarose Gels - Glassmilk Method

Elution of Small Fragments of DNA from Agarose Gels(<500bp)

Gel Purification of DNA from Agarose Gels by Centrifugation


POLYMERASE CHAIN REACTION (PCR)

Polymerase Chain Reaction

Melting Temperature Calculation


DNA MODIFYING ENZYMES

S1 Nuclease Digest of ssDNA

Alkaline Phosphatase Digestion of DNA

BAP

CIP


MANUAL SEQUENCING METHODS (ANCIENT HISTORY)

NEB Sequencing (Vent Polymerase)-- Preparing Samples to be Sequeneced

NEB Extension/Termination 35S-dATP Sequencing

Sequencing Gels

Sequencing Reaction: (Sequenase)


QUANTIFICATION

Fluorometer Procedures


USING OLIGONUCLEOTIDES

Melting Temperature Oligos

Phosphorylation with PNK


END REPAIR OF DNA FRAGMENTS

T4 Polymerase for Blunting 3' Extensions

Repair of Sticky Ends with Klenow (Large) Fragment of DNA Polymerase I

Mung Bean Nuclease Digestion


IN VIVO SYSTEMS

Competent Cells

Preparation of Competent E coli

DMSO Method

One Step Preparation

Transformation

Transformation of competent E coli

Efficiency

5 Minute Method


YEAST

Rapid Isolation

Rapid Isolation of Yeast Chromosomal DNA


TISSUE CULTURE

Aseptic Technique

Aseptic Technique

Grow and Store Cells

Passage or Subculturing Tissue Cells

Thawing Cells

Freezing Cells

Counting Cells

Fractionation

Fractionation of Tissue Culture Cells

Nitrogen Cavitation of Tissue Culture Cells

Phase Partitioning

P100 Membrane Prep from Tissue Culture Cells Method 1

P100 Membrane Prep from Tissue Culture Cells Method 2

Media and Reagents

Media and Reagents

Immunofluorescence

Immunofluorescence

Cytoskeleton

6A7 Bax Activation

Adenovirus

Adenovirus

Plaque Assays

Virus Shock Preparation

Infecting Cells with Adenovirus Ad5-strain

Transformation Assay

Focus Forming Assay

Semi Solid Media

Cell Lines

Established Cell Lines

Transfection

Transfection Expression Assay Method 1

Transfection Expression Assay Method 2

Picking Colonies

CaPO4 Precipitation

MDCK Cells

35S Labeling

35S Methionine Labeling

Apoptosis

Apoptosis Assay with MDCK cells

Mycoplasma Testing

Phoenix Cell Infection


MISC METHODS

GENERAL LAB PROCEDURES


PROTEIN CLEAVAGE

Hydroxylamine Cleavage of Asn-Gly


ISOELECTRIC FOCUSING

Isoelectric Focusing (IEF) of Membrane Proteins


TCA PRECIPITATION OF PROTEINS


CNBr

Coupling protein to CNBr activated Sepharose Beads


TRICINE SDS GELS

Preparation of Stock Solutions Required for Making Tricine SDS Gels

Preparation of Tricine SDS Gels

Preparation of the Stock Solutions Required for Running Tricine SDS Gels

Preparing to Run Tricine SDS Gels


QUANTITATIVE IMMUNOBLOT

Western Blotting

Preparing Samples for Western Blotting

Preparing Samples for Quantitative Western Blotting

Tricine SDS Gels

Electrophoretic separation

Electrophoretic Separation for Quantitative Western Blotting

Transfer

Quantitative Western Blotting

Developing Western Blots Using the ECL Method

Developing Western Blots with Scientific Imaging Film

Visualization of Western Blots on the Kodak Image Station


OVEREXPRESSION

OVEREXPRESSION INTRODUCTION


PRIT SYSTEMS

Purification of Fusion Protein on IgG Sepharose 6 FF

Induction of Temperature Sensitive Lambda Repressor


GST FUSIONS

The pGEX System


HIS TAGGED VECTORS

The pRSET System

Purification of His-Tag Proteins

Protocol for BID Protein Purification


PET VECTORS

The pET System


PrA VECTORS

Vectors: Protein A fusion


IMPACT SYSTEM


TOPP CELLS


TEMPERATURE INDUCTION OF LARGE SCALE BACTERIAL CULTURES


INCLUSION BODIES

Purification of Active Eukaryotic Proteins from the Inclusion Bodies in E.Coli

Purification of Proteins from Inclusion bodies

Promoter Variation for Avoiding Inclusion Bodies in Overexpression of Proteins

Cell Strain Variation for Avoiding Inclusion Bodies in Overexpression of Proteins

Sorbitol/Betaine Method for Avoiding Inclusion Bodies in Overexpression of Proteins


EXPRESSION TESTS

Preparing Total Cell Protein

How to Determine Whether Your Protein is Soluble or Insoluble


GLASS BEAD METHOD FOR ISOLATING TOTAL BACTERIAL PROTEINS


HARVEST 500mL

500ml Scale Production of Crude Bacterial Lysate

Large Scale Method: Sonication


INDUCTION PROTOCOL FOR TAC OVERECPRESSION VECTORS


PROTEIN ASSAYS

BCA Protein Assay (Pierce)

BioRad Protein Assay


RECIPES

DNA

CB 10X T4 DNA Ligase (+ATP)

Plasmid Preps

TE (Tris-EDTA Buffer)

BUFFERING PHENOL

ENZYME BUFFERS

DNA gel loading buffer (5 X)

(TBE) TRIS-BORATE

TBE DNA gel running buffer (Tris borate EDTA) 10 X

TRIS-ACETATE (TAE)

Transcription Recipes

IMMUNOLOGY

Buffers for immunoprecipitation

TRICINE GEL RECIPE

Preparation of Stock Solutions Required for Making Tricine SDS Gels

Preparation of Tricine SDS Gels

Preparation of the Stock Solutions Required for Running Tricine SDS Gels

Running Tricine SDS Gels


POLYACRYLAMIDE GELS

Laemmli Gels (SDS-PAGE)

Gel running/processing buffers

SDS-PAGE loading buffer

MICROSOMES

Microsomal Membranes From Dog Pancreas


MICROBIOLOGY

CaCl2 for Competent Bugs

Superbroth


RRL AND WG RECIPES

Retic Lysate Reagents

Retic Lysate More Reagents

Wheat Germ Buffers and Reagents

Wheat Germ Extract Recipes

WESTERN BLOTTING

Western Blotting

Western Blot Buffers