Morimoto Lab Protocols

1. Prokaryotic Cells

   1. Bacteriology

   2. Phage Preparation and Purification

      1. Bacteriophage preparation

      2. Packaging extracts for lambda cloning

      3. Bacteriophage M13

   3. Preparation of Competent Cells

      1. RbCl2 method

      2. Shawn's CaCl2

      3. DAM strains

   4. Transformation

      1. Optimizing DNA for transformation

      2. Transforming competent cells

      3. Transforming DH5 cells

   5. Library Screening

2. Eukaryotic Cells

   1. Cell Culture

      1. Introduction to cell culture

      2. Media preparation

      3. DMEM

      4. Dialysis of FBS

      5. Splitting

      6. Hemacytometer

      7. Coulter counter

      8. Trypan blue staining

      9. Freeze/thaw

      10. Freezing cell lines

      11. Thawing cells

      12. HeLa S3 cell culture

      13. Xenopus oocyte microinjection

      14. Preparation of extracts from human placenta

      15. Chicken blood injections

      16. Sacrifice of adult hen

      17. Bleeding eggs

      18. Chicken embryonic fibroblasts

   2. Cell Transfection

      1. CaPO4 method

      2. 3% CO2 method

      3. Ecogpt

      4. Trypsinization transfection

      5. Polyfect, Lipofectamine, and Effectene

      6. Cell sorting for transfected cells: MACs protocol

   3. Establishing Stable Cell Lines

      1. Calcium Chloride Transfection

      2. Lipofectamine Transfection (Gen's Protocol)

   4. Radiolabeling

      1. 32P orthophosphate and 35S methionine/cysteine

      2. Metabolic labeling

   5. Cell Growth/Death Assays

      1. Cell Death ELISA

      2. MTT

      3. TUNEL

      4. Drug Assays

   6. Luciferase Assays

      1. Luminometer operation

      2. Single luciferase

      3. Dual luciferase

      4. 96-well plate format

   7. RNAi - mammalian cells

   8. Cell Imaging

      1. Intro to immunofluorescence

      2. Method

      3. FRET

3. Protein Biochemistry

   1. Protein Expression and Purification

      1. Bacterial overexpression

      2. FPLC guidelines

      3. DEAE guidelines

      4. In-Vitro Translation

         1. General

         2. Nuclease Treatment of Rabbit Lysate

         3. Coupled translation/transcription

      5. Proteins

         1. Hsp70 columns

         2. Brian Freeman's hsp70

         3. Jaewan Song's hsp70

         4. Hip

         5. Hdj-1

         6. GST-Bag-1mm

         7. C. elegans CHIP

   2. Protein Analysis

      1. Electrophoresis

         1. SDS-PAGE (denaturing)

         2. Native SDS-PAGE/SOD active stain using native gel

         3. 2-dimensional

         4. Blatter gel

      2. Antibodies

         1. Table of antibodies

         2. Western blot analysis using ECL

         3. Semi-Dry Transfer

         4. Western Blot Analysis Using Alkaline Phosphatase

         5. Western Blot Using Licor Odyssey Analysis

         6. Immunoprecipation

            1. general

            2. HSF1 and HSF2

         7. Immunohistochemistry

      3. Bio-rad assay for quantification

   3. DNA-Protein Interactions

      1. Nuclear extracts

      2. Whole cell extracts

      3. Gel mobility shift analysis

      4. Super Shift

      5. 32P labeling of oligonucleotide probe

      6. In-vitro footprinting methods

      7. In-vivo footprinting methods

         1. Genomic DNA isolation

         2. Ligation mediated PCR

      8. ChIP

   4. Protein-Protein Interactions

      1. Glutaraldehyde and EGS crosslinking

      2. Equilibrium micro-dialysis

      3. GST-pulldown

   5. Filter Trap Assay for Aggregates

   6. Re-Folding Assays

      1. Brian Freeman's ATPase

      2. b-galactosidase

      3. In-vivo refolding

4. DNA Techniques

   1. Plasmid Preparation

      1. Mini prep

         1. Alkaline lysis method

         2. Boiling method

      2. Large prep

         1. Large scale

         2. Sue's large scale

         3. Differential precipitation

   2. Preparation of DNA from Cultured Cells

      1. High molecular weight

      2. Hirt method

      3. Total DNA extraction from mammalian cells

   3. DNA Electrophoresis

      1. Agarose - horizontal and vertical

      2. Polyacrylamide

      3. Alkaline agarose

   4. Cloning Techniques

      1. Fragment isolation

         1. Electroelution

         2. Acrylamide crush and soak

         3. Low melting point agarose

      2. Ligation

      3. Quick Ligation

   5. Sequencing

      1. Dideoxy (Sanger)

         1. Dideoxy sequencing with 35S

         2. Sequencing double-stranded DNA

         3. Dideoxy working solutions

         4. Automated dideoxy sequencing

      2. Maxam-Gilbert

      3. Big-Dye sequencing

   6. Modification

      1. Phosphatase treatment

         1. Calf intestinal phosphatase

         2. Bacterial alkaline phosphatase

      2. Kinase treatment

      3. Conversion of 5' ends to blunt ends

      4. 3' end filling

      5. Nick translation

      6. Restriction enzyme analysis

      7. Multiprime labeling

      8. Mutagenesis

   7. Amplification

      1. Polymerase chain reaction

      2. Quantitative RT-PCR

      3. Real-time PCR

      4. Colony PCR

5. RNA Techniques

   1. RNA Isolation

      1. Total RNA - GdSCN "Crude Susan"

      2. RNA from E. Coli cells

      3. Cytoplasmic RNA

      4. RNA (Trizol) extraction

   2. RNA Electrophoresis

      1. Glyoxal/PO4 agarose

      2. Formaldehyde

   3. Polysome Analysis of RNA

      1. Sucrose gradient

      2. Pelleted polysomes

6. Transcriptional Analysis

   1. Nuclear Run-On

   2. In-Vitro Transcription/Reverse Transcription

   3. SP6 Riboprobe System

   4. S1 Protection

   5. Primer Extention

   6. Hybrid Selection

   7. CAT Assay

   8. Luciferase Assay

   9. B-galactosidase Assay

7. Nucleic Acid Hybridization

   1. DNA/RNA Blotting and Hybridization

   2. Dot Blots

      1. DNA

      2. RNA

   3. Southern Blot Analysis

8. Yeast Methods:
   The Forsburg lab website is an excellent source of S. pombe information:
   Yast-Two-Hybrid Interaction Trap: See the protocols developed by the Brent lab

9. C. elegans Methods

   1. General

      1. Media and Solutions

      2. Freezing

      3. Bleaching/Synchronizing

      4. Generating males

      5. Mounting worms for imaging

      6. Preparing injection pads

   2. Extraction

      1. Genomic DNA

      2. Total RNA

      3. Whole cell extracts

   3. Staining

      1. Phalloidan

      2. Beta-galactosidase

      3. IF

      4. Amphid and phasmid staining

   4. Analyses

      1. Life-span

      2. Osmotic avoidance

      3. Osmotic (ors) stress

      4. Thrashing

   5. Genetics

      1. Integration by gamma irradiation

   6. RNAi

      1. Injection concentrations

      2. RNAi Plates

10. Appendix

1.      Common Stock Reagents

2.      Washing Glassware

3.      Washing Pipets

4.      Photography

5.      Densitometer

6.      Molecular Dynamics Phosphoimager