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Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4115

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR. A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems. The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization. - [Read Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium]

Chambers for Examination of Live Cells under Mechanical Stress Protocol - http://www.cshprotocols.org/cgi/content/extract/2007/3/pdb.ip32

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Manual measurement and manipulation of the cell surface requires access to the cells, usually in an open chamber. Temperature-controlled chambers or stage inserts are preferred for maintaining physiological activity during the experiment. For example, heated culture dishes with coverslip glass bottoms (Bioptechs) permit high-resolution fluorescence microscopy of living cells during force application. - [Read Chambers for Examination of Live Cells under Mechanical Stress Protocol]

Ethics, Guidelines for Animal Research PDF - http://www.brl.uic.edu/courses/TNotes05.pdf

Category: Animal Research Techniques

Needs of animals, Ventilation, lighting and temperature considerations. Cage size/density. Handling. Blood Collection and guidlines. EXCELLENT GUIDE GC470 ESSENTIALS FOR ANIMAL RESEARCH BRL Univ. Illinois. - [Read Ethics, Guidelines for Animal Research PDF]

Factors that Influence Restriction Enzyme Digestion Activity - http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/enzymes/cuteffects.html

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Factors that Influence Restriction Enzyme Digestion Activity. Buffer Composition, Incubation Temperature, DNA Methylation, Star Activity, Multiple Enzymes. Laura Austgen PhD et. al, Biomedical Hypertextbook, Colorado State Univ. - [Read Factors that Influence Restriction Enzyme Digestion Activity]

Fragmentation of DNA by Nebulization Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4539

Category: Cloning Protocols

Protocol describes a method for DNA fragmentation by nebulization, in which the fine mist created by forcing a DNA solution through a small hole in the nebulizer unit is collected. The size of the fragments obtained by nebulization is determined chiefly by the speed at which the DNA solution passes through the hole, altering the pressure of the gas blowing through the nebulizer, the viscosity of the solution, and the temperature. - [Read Fragmentation of DNA by Nebulization Protocol]

Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol - http://www.biologicalprocedures.com/bpo/arts/1/123/m123.pdf

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for the generation of gene deletions and gene replacements in Escherichia coli O157:H7 using a temperature sensitive allelic exchange system. Technology requires flanking DNA to be cloned into a temperature sensitive vector but the resulting clone allows great flexibility for further modification of the target sequence. It is therefore highly suited to the study of genes in which several rounds of changes are envisaged. - [Read Generation of Gene Deletions and Gene Replacements in Escherichia coli Protocol]

Hybridization of Oligonucleotide Probes in Aqueous Solutions Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3883

Category: Nucleic Acid Modification Protocols: Nucleic Acid Labeling Protocols

Hybridization is carried out in conventional aqueous solvents at a temperature well below the predicted melting temperature. Nonspecific hybrids are then removed by washing at high stringency in buffers containing quaternary salts. Tetramethylammonium chloride (TMACl) is used with probes that are 14-50 nucleotides in length, whereas tetraethylammonium chloride (TEACl) is used with longer oligonucleotides. - [Read Hybridization of Oligonucleotide Probes in Aqueous Solutions Protocol]

Indirect Peroxidase Technique Protocol - http://www.nottingham.ac.uk/pathology/protocols/indfroz.html

Category: Immunohistochemistry Protocols

Protocol for indirect peroxidase technique. Includes: Sections: Thickness-Between 5 and 10 microns. Pick up on chromic acid etched, poly-L-lysine or chrome alum/gelatine coated slides. Dry at room temperature overnight (not longer than 72 hours). - [Read Indirect Peroxidase Technique Protocol]

Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3929

Category: Cloning Protocols

Protocol for ligating plasmid and target DNAs in low-melting-temperature agarose. Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. - [Read Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol]

PCR and Multiplex Guide - http://info.med.yale.edu/genetics/ward/tavi/Guide.html

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Standard PCR Protocol

PCR and Multiplex Guide. Amazing Guide to primer amount, MgCl2 amount, annealing temperature, and more. With picture examples. Octavian Henegariu. - [Read PCR and Multiplex Guide]

PCR and Multiplex Guide - http://info.med.yale.edu/genetics/ward/tavi/Guide.html

Category: PCR Protocols: Standard PCR Protocol

PCR and Multiplex Guide. Amazing Guide to primer amount, MgCl2 amount, annealing temperature, and more. With picture examples. Octavian Henegariu. - [Read PCR and Multiplex Guide]

PCR primer design and pcr reaction optimization. - http://www.mcb.uct.ac.za/pcroptim.htm

Category: PCR Protocols: PCR Optimization

PCR primer design and pcr reaction optimization. Ed Rybicki. Factors Affecting the PCR, Denaturing Temperature and Time, Annealing Temperature and Primer Design, Primer Length, Degenerate Primers, Elongation , Temperature and Time, Reaction Buffer, Cycl - [Read PCR primer design and pcr reaction optimization.]

PCR Program Design - http://info.med.yale.edu/genetics/ward/tavi/p08.html

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: PCR Optimization Protocols

The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. P - [Read PCR Program Design]

PCR Program Design - http://info.med.yale.edu/genetics/ward/tavi/p08.html

Category: PCR Protocols: PCR Optimization

Phenol-based Protocol Isolation of DNA Fragments from Low-Melting Temperature Agarose - http://wheat.pw.usda.gov/~lazo/methods/101/101lmtagar.html

Category: DNA Protocols: DNA Extraction Protocols: Agarose Gel DNA Extraction Protocols

Phenol-based Protocol Isolation of DNA Fragments from Low-Melting Temperature Agarose. Reference: Favre, D. 1992. Biotechniques vol. 13. - [Read Phenol-based Protocol Isolation of DNA Fragments from Low-Melting Temperature Agarose]

Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3235

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

Protocol for preparation of DNA for pulsed-field gel electrophoresis: isolation of intact DNA from yeast. Yeast cells are first treated enzymatically to break down the cell walls and then resuspended in low-melting-temperature agarose plugs. The DNA is liberated by infusing the plugs with lysis buffer and proteases. This method is used to prepare both conventional and artificial yeast chromosomes. - [Read Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast]

Recovery of DNA from Low-melting-temperature Agarose Gels: Enzymatic Digestion with Agarase Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4026

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

A fragment of gel containing a band of DNA is excised and digested with agarase, which hydrolyzes the polymer to disaccharide subunits. The released DNA is then purified by phenol extraction and ethanol precipitation. The method works well for DNAs ranging in size from <5 kb to >20 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Enzymatic Digestion with Agarase Protocol]

Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4025

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

DNA fragments separated by electrophoresis through gels cast with low-melting-temperature agarose are recovered by melting the agarose and extracting the resulting solution with phenol:chloroform. The protocol works best for DNA fragments ranging in size from 0.5 kb to 5 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction Protocol]

Retrieval of DNA Fragments from Pulsed-field Gels following DNA Concentration Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4035

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]

Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3765

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

An expression library constructed in a bacteriophage {lambda} vector is plated on an appropriate E. coli strain in the absence of isopropylthio-ß-D-galactoside (IPTG). After 2-4 hours, the plates are moved to 37°C (to stabilize any fusion proteins that are temperature sensitive), and filters impregnated with IPTG are laid on top of the developing plaques. - [Read Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol]

Selection of Poly(A)+ RNA by Batch Chromatography - Subscription Required - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4048

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

When many RNA samples are to be processed or when working with small amounts (<50 Âµg) of total mammalian RNA, the technique of choice is batch chromatography on oligo(dT)-cellulose. The method described in this protocol uses a combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. Joseph Sambrook and David W. Russell. - [Read Selection of Poly(A)+ RNA by Batch Chromatography - Subscription Required]

Setting Up Microdrop Cultures Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4353

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Protocol describes how to set up microdrop cultures to produce embryos which can then be used for making chimeras. The microdrop culture should be set up several hours to 1 day before the experiment to permit temperature and gas equilibration. - [Read Setting Up Microdrop Cultures Protocol]

Transformation of E. Coli by Electroporation Protocol. - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3933

Category: Transfection Protocols: Electroporation Sonoporation Protocols

Electrocompetent bacteria are prepared by growing cultures tomid-log phase, washing the bacteria extensively at low temperature,and then resuspending them in a solution of low ionic strengthcontaining glycerol. DNA is introduced during exposure of thebacteria to a short high-voltage electrical discharge.

- [Read Transformation of E. Coli by Electroporation Protocol.]

Transposon Mediated Mutagenesis Protocol - http://www.genome.wisc.edu/resources/protocols/tnmutationprotocol.htm

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for transposon mediated mutagenesis. Includes: Amplify ORF from MG1655; Transposition reaction; Transformation; Picking; Verify mutation by Culture PCR; Confirm mutations by sequencing; Curing the temperature sensitive pKD46 plasmid. - [Read Transposon Mediated Mutagenesis Protocol]

Transposon-Mediated Mutagenesis Protocol - http://www.genome.wisc.edu/resources/protocols/tnmutationprotocol.htm

Category: Bacteria Genetics Protocols

Protocol for transposon-mediated mutagenesis. Includes: Amplify ORF from MG1655; Transposition reaction; Transformation; Picking; Verify mutation by Culture PCR; Confirm mutations by sequencing; Curing the temperature sensitive pKD46 plasmid. - [Read Transposon-Mediated Mutagenesis Protocol]

Articles (1-10 of 15)

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.

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Drosophila Cell Transfection Protocol

A simple Drosophila tissue culture cell transfection method.

Probe Preparation for 22 x 40 mm DNA Microarrays

Probe Preparation for 22 x 40 mm DNA Microarrays Protocol.

Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.

Paraffin Embedding Protocol

Paraffin Embedding Protocol for molecular profiling. This Paraffin Embedding Protocol describes the processing of the tissues into sections following ethanol fixation. Molecular profiling (MP) is a technique that is used to visualize the global patterns of RNA expression or protein expression in various cell types and disease processes.

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

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.