Dictyostelium Techniques (original) (raw)

Dictyostelium Techniques

Media and buffers | Growth and Development | Molecular Biology | Transformation | Mutagenesis | Gene Expression | Microscopy | Biochemistry | Cytoskeleton | Teaching Tools

Media and buffers

• Media and buffers
• Recipe for FM defined medium (contributed by Jakob Franke)
• Recipe for low fluorescence axenic medium (contributed by Harry MacWilliams)
• Recipe for synthetic medium (pdf) (contributed by Erwin Flaschel)

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Growth and development of Dictyostelium

• Methods for growing Dictyostelium
• Methods for_Dictyostelium_ development
• Dictyostelium cell storage procedures
• Plating Dictyostelium in soft agar *
• Methods for parasexual genetics (contributed by Jason King and Robert Insall)
• Method for determining mating types (contributed by Jakob Franke)

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Molecular Biology

• Quick extraction of genomic DNA (contributed by Steve Charette and Pierre Cosson)
• Isolation of genomic DNA *
• Isolation of genomic DNA with CsCl *
• RT-PCR for Knockout Screening and Expression Analysis (contributed by Robin Williams)

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Transformation

• Transformation protocols (Gaudet et al. 2007)
• Calcium phosphate precipitation *
• Electroporation (contributed by Dave Knecht)
• Transformation of NC4 or D. mucoroides (contributed by Harry MacWilliams)
• Microinjection (contributed by Yoshio Fukui)
• Transformation by particle gun (contributed by Birgit Wetterauer and Hans-Ulrich Koop)
• Addition of heat-killed bacteria to transformants *
• Transformant selection on bacterial lawns using the V18-Tn5-cassette (contributed by Harry MacWilliams)
• DAPI Electroporation

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Mutagenesis

• Restriction-enzyme mediated insertional mutagenesis (REMI)
• RNAi procedure (contributed by Alan Kimmel)
• Making double mutants by diploid genetics (contributed by Jason King and Robert Insall)

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Gene Expression

• Chromatin Immuno-precipitation (contributed by Jeff Williams and Y. Yamada)
• Whole mount in situ hybridization (contributed by Ricardo Escalante and Bill Loomis)
• Agar overlay technique (contributed by Yoshio Fukui)
• Indirect immunofluorescence *

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Microscopy

• Fixation techniques for immunonfluorescence (contributed by Richard Gomer)
• Visualizing weak fluorescence in multicellular stages (contributed by Harry MacWilliams)

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Biochemistry

• 35S-methionine labelling of Dictyostelium *
• 32phosphate labelling of Dictyostelium *

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Cytoskeleton

Protein preparation

• Isolation of Dictyostelium cytoskeleton *
• Isolation of Dictyostelium centrosomes (contributed by Ralph Gräf)
• Preparation of microtubule-associated motor proteins *
• Purification of muscle actin *
• One-day Dictyostelium myosin preparation *
• Three-day Dictyostelium myosin preparation *
• Dictyostelium cell staining for tubulin (contributed by Emma Dalton)

Protein assays

• Microtubule gliding assay for microtubule-associated motors *
• ATPase assay for dynein (radioactive) *
• CTPase assay for dynein (colorimetric) *
• in vitro motility assay *
• ATPase assay for Dictyostelium myosin *
• Estimation of the number of active myosin heads in isolated myosin *
• Myosin-F-actin binding by pelleting assay *

Protein detection

• Dictyostelium anti-γ-tubulin Westerns (contributed by Emma Dalton)

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Teaching Tools

* This experimental protocol was taken from the Rex Chisholm Lab's Dictyostelium Techniques Manual. Many of these protocols are the result of the contributions and collaborations of many different laboratories.