high Protocols

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]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

High-titer stocks of bacteriophage {lambda} are easily prepared by infecting small-scale bacterial cultures. - [Read Preparing Stocks of Bacteriophage {lambda} by Small-scale Liquid Culture Protocol]

Category: Protein Microarray Protocols

MacBeath G & Schreiber SL (2000). Printing proteins as microarrays for high-throughput function determination. Science 289: 1760-1763. MacBeath Lab - [Read Printing proteins as microarrays for high-throughput function determination.]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Procedures for labeling DNA, RNA, and oligonucleotides with DIG, biotin, or fluorochromes. Includes: Random primed labeling of ds DNA with DIG-, Biotin- or Fluorescein-High Prime reaction mix; PCR labeling of ds DNA with the PCR DIG Probe Synthesis Kit or PCR Labeling Mixes. - [Read Procedures for Labeling DNA, RNA, and Oligonucleotides with DIG, Biotin, or Fluorochromes]

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Procedures for labeling DNA, RNA, and oligonucleotides with DIG, Biotin, or Fluorochromes. Includes: Random primed labeling of ds DNA with DIG-, Biotinor Fluorescein-High Prime reaction mix; PCR labeling of ds DNA with the PCR DIG Probe Synthesis Kit or PCR Labeling Mixes. - [Read Procedures for Labeling DNA, RNA, and Oligonucleotides with DIG, Biotin, or Fluorochromes]

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]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA in solid tissue samples using either propidium iodide (PI) or DAPI staining followed by flow cytometry. PI can be excited at 488 nm by the argon-ion laser, the most commonly used laser in flow cytometry. Alternatively, DAPI is best excited by a high-power UV laser, which is less commonly available. - [Read Propidium Iodide (PI) or DAPI Staining of Unfixed Solid Tissues for Flow Cytometry Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA in tissue culture cells using either propidium iodide (PI) or DAPI staining followed by flow cytometry. PI can be excited at 488 nm by the argon-ion laser, the most commonly used laser in flow cytometry. Alternatively, DAPI is best excited by a high-power UV laser, which is less commonly available. - [Read Propidium Iodide (PI) or DAPI Staining of Unfixed Tissue Culture Cells for Flow Cytometry Protocol]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol describes the separation of proteins using ceramic hydroxyapatite and HPLC. - [Read Protein Separation on Ceramic Hydroxyapatite Using High-Performance Liquid Chromatography Protocol]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Includes protocols: Direct Adaptation to HyQ© SFX-Insect™ Medium; Sequential Adaptation to HyQ© SFX-Insect™ Medium; Adaptation of High Five™ Cells to HyQ© SFX-Insect™; Production of Recombinant Protein Using the Baculovirus Expression Vector System; Culturing Insect Cells and Production of Baculovirus Expressed Proteins in 2.8 L Fernbach Systems. Production of Autographa californica Multiple Capsid Nuclear Polyhedrosis Virus (rAcMNPV) Stock - [Read Protocols for Insect Cell Culture]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Labeling Protocols

Protocol for purification of labeled probes using the High Pure PCR Product Purification Kit. - [Read Purification of Labeled Probes Using the High Pure PCR Product Purification Kit Protocol]

Category: Nucleic Acid Purification Protocols

Protocol for purification of labeled probes using the High Pure PCR Product Purification Kit. - [Read Purification of Labeled Probes using the High Pure PCR Product Purification Kit Protocol]

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

Peroxisomes can be purified in self-generated iodixanol gradients in high yield (80-90%) with no detectable contamination from any other organelle. In iodixanol peroxisomes are the densest of the major subcellular organelles (ρ = 1.18-1.20 g/ml) present in the light mitochondrial fraction from mammalian tissues and cells. - [Read Purification of Peroxisomes in a Self-Generated Gradient]

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

Peroxisomes can be purified in iodixanol gradients in high yield (80-90%) with no detectable contamination from any other organelle. This is a property unique to iodixanol because the densities of other organelles, particularly that of mitochondria (approx ρ = 1.14 g/ml) and endoplasmic reticulum (approx ρ = 1.13 g/ml) are much lower than that of peroxisomes (approx ρ = 1.18 g/ml). - [Read Purification of Peroxisomes using a Density Barrier in a Swinging-Bucket Rotor]

Category: Cell Biology and Cell Culture

Protocol for the recovery of a highly viable fraction for use in fertilization. Protocol uses an iodinated density gradient medium to adjust the density of the raw ejaculate to approx 1.170 g/ml by mixing with a high density medium (ρ >1.26 g/ml) and loading it beneath a discontinuous gradient. - [Read Purification of viable bovine spermatozoa of normal morphology.]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

Recovery of bands of DNA from agarose gels by electrophoresis onto a sliver of DEAE-cellulose membrane can be performed simultaneously on many samples and reliably gives high yields of fragments between 500 bp and 5 kb in length. - [Read Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Plasmid Preps Protocols

High-molecular-weight RNA and proteins can be precipitated from preparations of plasmid DNA by high concentrations of LiCl and removed by low-speed centrifugation. - [Read Removal of Small Fragments of Nucleic Acid from Preparations of Plasmid DNA by Precipitation with Li]

Category: Immunohistochemistry Protocols: Resin Staining Protocols

Protocol for resin embedding for high resolution morphology. - [Read Resin Embedding for High Resolution Morphology Protocol]

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol for restriction digests of high molecular weight yeast DNA. - [Read Restriction Digests of High Molecular Weight Yeast DNA Protocol]

Category: Nucleic Acid Detection Protocols

Restriction landmark genomic scanning (RLGS) is a method to detect large numbers of restriction landmarks in a single experiment.  It is based on the concept that restriction enzyme sites can serve as landmarks throughout a genome.  RLGS uses direct end-labeling of the genomic DNA digested with a rare-cutting restriction enzyme and high-resolution two-dimensional electrophoresis. - [Read Restriction Landmark Genomic Scanning Protocol]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for retrieval of DNA fragments from pulsed-field gels following DNA concentration. DNA contained in a slice of low-melting-temperature agarose is first concentrated by electrophoresis into a high-percentage agarose gel, and then isolated by treatment with agarase.  The resulting DNA preparation is purified by microdialysis. - [Read Retrieval of DNA Fragments from Pulsed-field Gels following DNA Concentration Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol exploits the discovery that Rnase A can efficiently cleave at single rC or rU bases embedded in double-stranded DNA.  Entire plasmid vectors are amplified using long, high-fidelity PCR with riboprimers, which carry a single rC residue at their 3' end.  Target DNA is amplified using similar primers, which also end in a rC residue. - [Read Ribocloning: DNA Cloning and Gene Construction Using PCR Primers Terminated with a Ribonucleotide]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

In vitro transcription systems includes instructions for use of products P1420, P1430, P1440, P1450 and P1460.Includes information and protocols on RNA Transcription in vitro. Information on DNA Template Preparation;Synthesis of High-Specific-Activity Radio labeled RNA Probes;Determining Percent Incorporation and Probe Specific Activity;Removal of the DNA Template Following Transcription;Removal of unincorporated nucleotides;Synthesis of large amounts of RNA;Capping RNA for in vitro translation. - [Read Riboprobe In Vitro Transcription Systems]

Category: Cytogenetics Protocols: RNA In Situ Hybridization Protocols

RNA-RNA in situ hybridization using DIG-labeled probes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. RNA-RNA in situ hybridization protocol using alkaline phosphatase-conjugated digoxigenin-(DIG-) labeled probes is presented. The addition of polyvinyl alcohol (PVA) of high molecular weight (40 – 100 kD) to the BCIP-NBT detection system enhances the alkaline phosphatase reaction and prevents... - [Read RNA-RNA In Situ Hybridization Using DIG-Labeled Probes Protocol]