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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]

DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4098

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

In this protocol, the DNA-binding capacity of Wizard MagneSil particles is used to capture and release a consistent amount of DNA (100 ng) across a wide range of samples. At the end of the procedure, the DNA is eluted into 100 µl Elution Buffer to give a final concentration of 1 ng/µl, relieving the need for postpurification DNA quantitation. - [Read DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs Protocol]

DNA Transfection by Biolistics Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3874

Category: Gene Delivery Protocols: Biolistics Protocols

In this protocol, gold or tungsten particles are coated with DNA and then shot from a gun into monolayers of mammalian cells. - [Read DNA Transfection by Biolistics Protocol]

Dye Terminator Cleanup for Automated DNA Sequencing Reactions Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4101

Category: DNA Protocols: DNA Sequencing Protocols

Protocol describes a simple procedure for purifying sequencing reactions using the MagneSil particles from Promega. - [Read Dye Terminator Cleanup for Automated DNA Sequencing Reactions Protocol]

Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Proteinase K and SDS Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3972

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

DNA is best isolated from bacteriophage lambda particles by digesting the viral coat proteins with a powerful protease such as proteinase K, followed by extraction with phenol:chloroform. This procedure is easily adapted for use with smaller-scale preparations of bacteriophage lambda. - [Read Extraction of Bacteriophage lambda DNA from Large-scale Cultures Using Proteinase K and SDS Protocol]

Fractionation of Mitochondria, Lysosomes, Peroxisomes, ER and Golgi in Pre-formed Iodixanol Gradient - http://www.axis-shield.com/densityhome/optiprep/S13.pdf

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Mitochondria Isolation Protocols

Generally in iodixanol gradients the density of organelles decreases in the series: peroxisomes, mitochondria, lysosomes, ER, Golgi, although in Dictyostelium discoideum, the lysosomes are denser than the mitochondria. Iodixanol gradients can usually provide satisfactory resolution of all these membrane particles although it may be necessary to modulate either the gradient or centrifugation parameters in order to optimize a particular separation. - [Read Fractionation of Mitochondria, Lysosomes, Peroxisomes, ER and Golgi in Pre-formed Iodixanol Gradient]

Gene Delivery to Skin Using Biolistics Protocol - http://www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4446

Category: Gene Delivery Protocols: Biolistics Protocols

The technique has many advantages—plasmids may be used for delivery, DNA theoretically can be delivered to any cell type, and genes may be delivered to cells in vitro, ex vivo, or in vivo. DNA-coated gold particles are distributed evenly along the length of the tubing, which is subsequently cut into short sections of cartridges to be used in a gene gun. The Helios Gene Gun uses a pulse of helium to launch the DNA-coated particles, spreading them onto the target cells. - [Read Gene Delivery to Skin Using Biolistics 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]

Precipitation of Bacteriophage {lambda} Particles from Large-scale Lysates Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3966

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Bacteriophage {lambda} particles are recovered from bacterial lysates by precipitation with polyethylene glycol at high ionic strength. - [Read Precipitation of Bacteriophage {lambda} Particles from Large-scale Lysates Protocol]

Preparation of Crude Subcellular Fractions by Differential Centrifugation Protocol - http://www.axis-shield.com/densityhome/optiprep/S07.pdf

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

The employment of differential centrifugation to prepare crude fractions of subcellular particles from homogenates is often a necessary first step to a subsequent purification of one or more particles on a density gradient. This protocol describes the use of differential centrifugation to fractionate a mammalian liver homogenate but similar methods should be applicable to all mammalian tissues and cultured cells. - [Read Preparation of Crude Subcellular Fractions by Differential Centrifugation Protocol]

Preparation of Single-stranded Bacteriophage M13 DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3686

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Bacteriophage M13 single-stranded DNA is prepared from virus particles secreted by infected cells into the surrounding medium. The filamentous particles are concentrated by precipitation from a high-ionic-strength buffer with polyethylene glycol. Subsequent extraction with phenol releases the single-stranded DNA, which is then collected by precipitation with ethanol. This protocol is generally used to prepare single-stranded DNA from a small number of M13 isolates. - [Read Preparation of Single-stranded Bacteriophage M13 DNA Protocol]

Producing Single-stranded DNA with Phagemid Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4019

Category: Viral Manipulation Protocols: Phage Protocols

Protocol describes methods to superinfect bacteria carrying a recombinant phagemid with a high-titer stock of an appropriate helper virus and to assay the yield of filamentous virus particles that carry single-stranded copies of the phagemid DNA. The key to success in using phagemids is to prepare a stock of helper virus whose titer is accurately known. - [Read Producing Single-stranded DNA with Phagemid Vectors Protocol]

Purification of Bacteriophage lambda Particles by Isopycnic Centrifugation through CsCl Gradients - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3968

Category: Viral Protocols

Isopycnic centrifugation through CsCl gradients is used to prepare infectious bacteriophage {lambda} particles of the highest purity that are essentially free of contaminating bacterial nucleic acids. - [Read Purification of Bacteriophage lambda Particles by Isopycnic Centrifugation through CsCl Gradients]

Purification of Oocyst Walls and Sporocysts from Toxoplasma gondii Protocol - http://www.axis-shield.com/densityhome/optiprep/C34.pdf

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Although Percoll gradients were able to provide a purified sporocyst fraction, because these particles do not all band in a discrete manner in such gradients, they were unable to provide a simultaneous isolation of a pure oocyst wall fraction. Gradients formed from this protocol on the other hand are able to provide purified sporocysts and oocyst walls in the same gradient. - [Read Purification of Oocyst Walls and Sporocysts from Toxoplasma gondii Protocol]

Screening a cDNA Library for use with HybriZAP zebrafish cDNA libraries - http://www.cbs.umn.edu/~kclark/protocols/library.html

Category: RNA Protocols: cDNA Libraries Library

Procedures: Titer the library to determine the concentration of infectious phage, Plate and lift the phage particles, Hybridize the immobilized cDNA with labeled probe, Pick positive plaques. Karl Clark U. Minn. - [Read Screening a cDNA Library for use with HybriZAP zebrafish cDNA libraries]

Subfractionation of Low-Density Lipoprotein (LDL) - http://www.axis-shield.com/densityhome/optiprep/M11.pdf

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

In the routine method described in this protocol, chylomicron-free plasma is adjusted to 12% (w/v) iodixanol and the sample, essentially fills an approx 3 ml tube for a near-vertical rotor. During the centrifugation VLDL, LDL and HDL particles and also plasma proteins migrate from all parts of the sample to their final buoyant density banding position in the self-forming density gradient. - [Read Subfractionation of Low-Density Lipoprotein (LDL)]

Virion RNA Dot Blot Protocol - http://www.bio.com/protocolstools/protocol.jhtml?id=p617

Category: Viral Protocols

Protocol describes the production of a dot blot membrane spotted with RNA isolated from virus particles. - [Read Virion RNA Dot Blot Protocol]

Articles (1-4 of 4)

Single Stranded Plasmid DNA Isolation Protocol

A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.

DNA Fragment Purification from Polyacrylamide Gels Protocol

A simple protocol for the purification of DNA fragments from Polyacrylamide Gels.

Absorption Spectroscopy and Quantification of Filamentous Phage Protocol

Unlike spherical phage, such as T4 and λ, which have roughly equal weight ratios of protein to DNA, filamentous phage have about six times more protein than DNA; the protein therefore contributes substantially to the absorption spectrum.

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