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Calibration of Micropipette Tips Protocols - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4655

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes an easy method for calibrating micropipette tips that have been pulled in the laboratory. It is essential to estimate the internal diameter of the pulled micropipette tip when adjusting parameters for a new puller or new type of glass tubing. A tip diameter of ~0.3 µm is optimal for the microinjection of mammalian cells in culture (e.g., CHO, PtK1, and COS-7). A 10% increase in diameter increases the delivery rate by more than 30% and can cause cell damage. - [Read Calibration of Micropipette Tips Protocols]

Collecting Two-Cell- to Compacted Morula-Stage Embryos Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4359

Category: Stem Cell Protocols: Stem Cell Injection Protocols

This protocol describes a method for collecting two-cell- to compacted morula-stage embryos. Such embryos can then be used for microinjection. - [Read Collecting Two-Cell- to Compacted Morula-Stage Embryos Protocol]

Collecting Zygotes and Removing Cumulus Cells With Hyaluronidase Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4358

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for collecting zygotes from pregnant female mice. The zygotes can then be used for microinjection. - [Read Collecting Zygotes and Removing Cumulus Cells With Hyaluronidase Protocol]

Delivery of dsRNA into Drosophila Embryos by Gene Gun Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4517

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how subcellular-sized particles are accelerated to high velocity to carry double-stranded RNA (dsRNA) into Drosophila embryos. The major advantage of this procedure over microinjection (Microinjection of dsRNA into Drosophila Embryos) is that particle bombardment is easier and faster to perform. In addition, the mechanical trauma received is far less than by microinjection, allowing better survival of embryos and fewer phenotypic artifacts. - [Read Delivery of dsRNA into Drosophila Embryos by Gene Gun Protocol]

Differences in Injection of X. laevis and X. tropicalis - http://tropicalis.berkeley.edu/home/manipulate_embryos/microinjection/differences.html

Category: Xenopus Protocols: Xenopus Injection Protocols

Differences in injection of X. laevis and X. tropicalis. Includes: X. tropicalis lays eggs about 4 hours after a boost of hCG; In vitro fertilization is not nearly as efficient in trops compared to laevis; X. tropicalis embryos are much softer than X. laevis embryos; X. tropicalis embryos whose jelly coats are removed by cysteine have a loose sticky vitelline membrane; X. tropicalis do not have a "summer slump". - [Read Differences in Injection of X. laevis and X. tropicalis]

DNA Preparation and Needle Loading for Gene Delivery by Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4652

Category: Microinjection Protocols: Microinjection of DNA Protocols

For microinjection into cells, high-quality plasmid DNA is purified using standard procedures. The resulting preparation is then delivered into the microinjection needle by either a back-filling or a forward-filling approach. - [Read DNA Preparation and Needle Loading for Gene Delivery by Microinjection Protocol]

Drosophila Embryo Microinjection Protocol - http://vosshall.rockefeller.edu/protocols/DrosophilaEmbryo.pdf

Category: Drosophila Protocols

Protocol for Drosophila embryo microinjection. - [Read Drosophila Embryo Microinjection Protocol]

Gene Delivery by Direct Injection (Microinjection) Using a Controlled-Flow System Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4654

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for constant-flow microinjection using the Pneumatic PicoPump (World Precision Instruments). This type of system is very simple and can be assembled on a relatively low budget. In this method, a constant flow of sample is delivered from the tip of the pipette, and the amount of sample injected into the cell is determined by how long the pipette remains in the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Controlled-Flow System Protocol]

Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/30/pdb.prot4653

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol]

Genetically-Engineered Mouse Models - http://www.bcm.edu/dtmc/

Category: Microinjection Protocols: Microinjection of DNA Protocols

Microinjection Services – mouse ES cells into blastocysts and DNA into pronucleus of fertilized eggs. - [Read Genetically-Engineered Mouse Models]

Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4673

Category: Reporter Gene Assays: GFP Assay Protocols

Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4673

Category: Microinjection Protocols

Fluorescence microscopy provides a powerful tool for imaging molecular components in living cells. Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Introduction of Caged Peptide/Protein into Cells Using Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4659

Category: Microinjection Protocols: Microinjection of Proteins Protocols

Activation and inactivation of proteins using photoactivation of caged peptides or proteins offer insights into cellular dynamics not achievable using genetic means. The ability to selectively alter the activity of a specific protein at a defined time and location inside a cell allows the correlation of changes in protein activity and cellular behavior. A caged compound, peptide, or protein is prepared by covalently linking it to a photolabile, protecting group. - [Read Introduction of Caged Peptide/Protein into Cells Using Microinjection Protocol]

Introduction of Double-Stranded RNA in C. elegans by Injection Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4315

Category: C. elegans Protocols: C. elegans Genetics Protocols

Double-stranded RNA (dsRNA) can be efficiently introduced into Caenorhabditis elegans by microinjection into the gonad, the gut, or the body fluid. The RNAi effect will spread within the nematode, exerting an effect beyond the site of injection. - [Read Introduction of Double-Stranded RNA in C. elegans by Injection Protocol]

Introduction of Double-Stranded RNA in C. elegans by Injection Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4315

Category: RNAi Technology Protocols: RNAi C. elegans Protocols

Large-Scale Preparation of Agarose Plugs of Yeast DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4392

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes the use of agarose plugs for isolation of yeast artificial chromosome (YAC) DNA. The DNA can then be run on a pulsed-field gel and used for microinjection to produce transgenic mice. - [Read Large-Scale Preparation of Agarose Plugs of Yeast DNA Protocol]

Making Holding Pipettes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4394

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes a method for making holding pipettes, to be used for microinjection. - [Read Making Holding Pipettes Protocol]

Making Holding Pipettes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4394

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes a method for making holding pipettes, to be used for microinjection. - [Read Making Holding Pipettes Protocol]

Making Injection Pipettes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4395

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes the determination of useful settings with the Sutter Puller P-97 to make injection pipettes for microinjection. - [Read Making Injection Pipettes Protocol]

Making Injection Pipettes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4395

Category: Mouse Protocols: Mouse Embryology Protocols

Protocol describes the determination of useful settings with the Sutter Puller P-97 to make injection pipettes for microinjection. - [Read Making Injection Pipettes Protocol]

Microinjection of DNA into Pronucleus of Fertilized Eggs Protocol - http://www.bcm.edu/dtmc/?pmid=7785

Category: Microinjection Protocols: Microinjection of DNA Protocols

Protocol for microinjection of DNA into pronucleus of fertilized eggs. - [Read Microinjection of DNA into Pronucleus of Fertilized Eggs Protocol]

Microinjection of dsRNA into Drosophila Embryos Protocol - http://www.cshprotocols.org/cgi/content/full/2006/21/pdb.prot4516

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol provides a description of how to introduce double-stranded RNA (dsRNA) into Drosophila embryos by microinjection. Several days of preparation are required before injections into Drosophila embryos begin. Flies must be in abundant supply for egg collection. Bombardment of embryos with dsRNA-coated gold particles (Delivery of dsRNA into Drosophila Embryos by a Gene Gun) can be used as an alternative. - [Read Microinjection of dsRNA into Drosophila Embryos Protocol]

Microinjection of dsRNA into Mouse Oocytes and Early Embryos Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4511

Category: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes how to introduce a double-stranded RNA (dsRNA) of choice into mouse oocytes or fertilized one-cell embryos by microinjection. For collection of mouse oocytes and early embryos, see Collection of Mouse Oocytes for RNAi and Collection of Early Mouse Embryos for RNAi. - [Read Microinjection of dsRNA into Mouse Oocytes and Early Embryos Protocol]

Microinjection of Mouse ES Cells into Blastocysts - http://www.bcm.edu/dtmc/?pmid=7791

Category: Microinjection Protocols: Microinjection of DNA Protocols

The core defines one injection as a total of forty blastocysts injected on two consecutive days using one or two clones for the same mutation. Please, refer to the Pricing page for information on the cost of one injection. - [Read Microinjection of Mouse ES Cells into Blastocysts]

Microinjection of Mouse Zygotes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4397

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for microinjection of mouse zygotes to produce transgenic mice. - [Read Microinjection of Mouse Zygotes Protocol]

Articles (1-4 of 4)

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

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