Bowtell Lab manual v31.8.95.

The following set of protocols were contributed by various members of our lab (past and present):

Christine Andrews, Fiona Christensen, Neil Della, Debbie Donald, Caroline Fernandez, Andrew Holloway, Yinling Hu, Maria Kafali, Rachael Parkinson, Hannah Robertson and Dennis Wang. Special thanks to Vicki Hammond who contributed the bulk of the ES cell protocols. Sections dealing with photomicroscopy, polyclonal and monoclonal antibody production were provided by members of Gerry Rubin's laboratory (Berkeley).

Any comments in the methods (technical errors etc.) email: bowtell@ariel.ucs.unimelb.edu.au

David Bowtell PMCI August 1995

Contents

• Isolation of DNA fragments using glass milk (GENE-CLEAN)
• SOUTHERN BLOT
• SOUTHERN ANALYSIS OF MOUSE TOE/TAIL DNA
• Northern transfer protocol
• Hybridizing Northern blots with riboprobes
• cDNA + GENOMIC DNA LIBRARY SCREENING
• Isolation of Mouse genomic DNA
• RNA ISOLATION USING GUHCL
• POLY A+ RNA PREPARATION using proteinase K and SDS
• SELECTION OF POLYADENYLATED RNA FROM TOTAL RNA
• Mini plasmid preps
• Large Scale Plasmid Preps
• Preparing and USING bacterial cells for electroporation
• In situ hybridisation to RNA in tissue sections
• PRIMER LABELLING FOR PRIMER EXTENSION ASSAYand PRIMER EXTENSION
• DNA-se I FOOTPRINTING
• Double stranded DNA sequencing (Dideoxy)
• Buffer gradient SEQUENCING GELS
• Nested Deletions using exonuclease-III and mung bean nuclease
• SITE DIRECTED MUTAGENESIS USING THE UNG - DUT SYSTEM.
• In vitro mutagenesis using Altered Sites

• Labeling of oligonucleotides
  • RANDOM HEXAMER LABELLING OF CDNA PROBES
  • RNAse protection assay
  • Isolation of phage DNA
  • PHAGE PURIFICATION WITH DE52
  • BUTANOL PRECIPITATION OF DEPROTECTED OLIGONUCLEOTIDES
  • OLIGO PURIFICATION on acrylamide gels
  • POLYMERASE CHAIN REACTION
  • Rapid Amplification of mRNA ENDS by PCR (RACE)
  • YEAST MEDIA
  • TRANSFORMATION OF YEAST
  • Modified yeast transformation
  • Isolation of shuttle vectors from yeast and transformation into E. coli
  • Yeast lysates for westerns

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