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Analysis of Recombinant Bacteriophage M13 Clones Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3996

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

A rapid method to analyze the size of the single-stranded DNA of M13 recombinants. - [Read Analysis of Recombinant Bacteriophage M13 Clones Protocol]

Attaching Adaptors to Protruding Termini Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3920

Category: Cloning Protocols

Adaptors are short double-stranded synthetic oligonucleotides that carry an internal restriction endonuclease recognition site and single-stranded tails at one or both ends. Adaptors are used to exchange restriction sites at the termini of linear DNA molecules. They may be purchased in phosphorylated and unphosphorylated forms. - [Read Attaching Adaptors to Protruding Termini Protocol]

Attachment of Linkers or Adaptors for Construction of cDNA Libraries - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3310

Category: DNA Protocols: cDNA Library Protocols

This stage achieves four goals: polishing the ends of double-stranded DNA, ligation of synthetic linkers or adaptors, digestion of the attached linkers to create cohesive termini, and preparing the cDNA for cloning. - [Read Attachment of Linkers or Adaptors for Construction of cDNA Libraries]

Choosing the Right Labeling Method for your Hybridization Experiment - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_15-18.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Choosing the right labeling method for your hybridization experiment. Includes: Homogeneous labeling methods for DNA; Homogeneous labeling methods for RNA; Stability of probe-target interaction; Nonradioactive labeling of oligonucleotides; Double-stranded versus single-stranded probes. - [Read Choosing the Right Labeling Method for your Hybridization Experiment]

Collection of Early Mouse Embryos for RNAi Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4510

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

Protocol describes a method to collect early embryos from 6-week-old mice. Subsequently, the isolated embryos can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Early Mouse Embryos for RNAi Protocol]

Collection of Mouse Oocytes for RNAi Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4509

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

Protocol describes a method to collect oocytes from 6-week-old mice. Subsequently, the isolated oocytes can be injected with double-stranded RNA to induce knockdown of a gene of interest. - [Read Collection of Mouse Oocytes for RNAi Protocol]

Competent cell preparation - http://www.chem.uga.edu/scottgrp/GrpProtocols/Competent_cell_preparation.htm

Category: Microbiology: Bacterial Competent Cells Preparation E.Coli

Bacterial E.Coli competent cell preparation. Calcium Chloride Protocol, Preparation of calcium chloride competent cells for frozen storage, Electroporation Protocol, Electroporation Protocol for transformations using double-stranded plasmids, Reference: - [Read Competent cell preparation]

Construction of cDNA Libraries Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3560

Category: DNA Protocols: cDNA Library Protocols

Here, the DNA-RNA hybrids synthesized in Stage 1 are converted into full-length double-stranded cDNAs. The primers for synthesis of second-strand cDNA are created by RNase H, which introduces nicks into the RNA moiety of the cDNA-mRNA hybrids. E. coli DNA polymerase I extends the newly created 3'-hydroxyl termini, using the first-strand cDNA as a template. - [Read Construction of cDNA Libraries Protocol]

Construction of cDNA Libraries Protocol. - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4073

Category: DNA Protocols: cDNA Library Protocols

The goal of this stage is to introduce methyl groups that will modify and protect naturally occurring EcoRI sites in the double-stranded cDNA. - [Read Construction of cDNA Libraries Protocol.]

Construction of the Sequencing Library Protocol - http://sequence-www.stanford.edu/protocols/library.html

Category: DNA Protocols: DNA Sequencing Protocols

Protocol first describes the vector preparation and, then, describes the insert preparation. Vital to have an excellent vector in order to produce a sequencing library. Protocol employs the male-specific coliphage M13 as the sequencing vector. M13 is a filamentous phage with a single-stranded, circular genome. M13 is widely used as a vector because many versions are available commercially and because M13 has certain advantages. - [Read Construction of the Sequencing Library 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]

Detection of DNA in Agarose Gels Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4022

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

The most convenient and commonly used method to visualize DNA in agarose gels is staining with the fluorescent dye ethidium bromide. Ethidium bromide can be used to detect both singleand double-stranded nucleic acids (both DNA and RNA). However, the affinity of the dye for single-stranded nucleic acid is relatively low and the fluorescent yield is comparatively poor. - [Read Detection of DNA in Agarose Gels Protocol]

Detection of Mutations by Single-strand Conformational Polymorphism Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3815

Category: Genetics and Genomics: Cancer Genetics Protocols: Single Strand Conformational Polymorphism(SSCP) Protocols

Single-strand conformational polymorphism (SSCP), one of several methods used to scan segments of DNA for mutations, exploits the electrophoretic differences in mobilities between single-stranded mutant and wild-type DNAs. - [Read Detection of Mutations by Single-strand Conformational Polymorphism Protocol]

Dideoxy-mediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase (Sequenase) Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3801

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol describes DNA-sequencing of single-stranded DNAtemplates in reactions catalyzed by Sequenase. - [Read Dideoxy-mediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase (Sequenase) Protocol]

Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3791

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

In this protocol asymmetric PCR is used to generate single-strandedDNA templates for dideoxy-sequencing - [Read Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers Protocol]

Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3802

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol for dideoxy-mediated sequencing reactions using the Klenow fragment of E. coli DNA polymerase I and single-stranded DNA templates. - [Read Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase]

Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase I - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3802

Category: E Coli Protocols: E coli Nucleic Acid Protocols

Protocol for dideoxy-mediated sequencing reactions using the Klenow Fragment of E. coli DNA polymerase I and single-stranded DNA templates. - [Read Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase I]

Electrophoresis - Agarose Gels for Single Stranded DNA Protocol - http://rothlab.ucdavis.edu/protocols/E02.html

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

Protocol for electrophoresis of single stranded DNA using agarose gel. - [Read Electrophoresis - Agarose Gels for Single Stranded DNA Protocol]

End Labeling Protruding 3' Termini of Double-stranded DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3853

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

Protocol for end labeling protruding 3' termini of double-stranded DNA with [{alpha}-32P]Cordycepin 5'-Triphosphate or [{alpha}-32P]dideoxyATP. - [Read End Labeling Protruding 3' Termini of Double-stranded DNA Protocol]

FIXATION and DNA Staining for Cell Cycle Analysis - http://www.biotech.iastate.edu/facilities/CELLHYB/CCAnalysis.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

FIXATION and DNA Staining for Cell Cycle Analysis Protocol. This method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane, which allows the dye (Propidium Iodide) to enter the cells. Propidium Iodide (PI) is a DNA-binding fluorochrome that intercalates in the double-helix. Ribonuclease-A is used to eliminate the staining of double-stranded RNA. - [Read FIXATION and DNA Staining for Cell Cycle Analysis]

Fluorometric Quantitation of DNA Using Hoechst 33258 Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4458

Category: Nucleic Acid Detection Protocols

Protocol describes the quantitation of DNA using Hoechst 33258, a fluorescent dye that binds to double-stranded DNA. Fluorometry is simple and more sensitive than spectrophotometry, and allows the detection of nanogram quantities of DNA. The assay can only be used to measure the concentration of DNAs whose sizes exceed ~1 kb, as Hoechst 33258 binds poorly to smaller DNA fragments. - [Read Fluorometric Quantitation of DNA Using Hoechst 33258 Protocol]

Generation of Bidirectional Sets of Deletion Mutants by Digestion with BAL 31 Nuclease Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3589

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

In this method, the nuclease BAL 31 is used to make uni- or bidirectional deletions in a segment of cloned DNA. BAL 31 is a complex enzyme and tends to digest a population of double-stranded DNA targets in an asynchronous fashion, Deletions created by BAL 31 are therefore far more heterogeneous in size than those created by processive enzymes such as exonuclease III. - [Read Generation of Bidirectional Sets of Deletion Mutants by Digestion with BAL 31 Nuclease Protocol]

Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4078

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The double-stranded DNA of recombinant plasmid, phagemid, or bacteriophage M13 replicative form DNA is digested with two restriction enzymes whose sites of cleavage both lie between one end of the target DNA and the binding site for universal primer. The enzyme that cleaves nearer the target sequence must generate either a blunt end or a recessed 3' terminus; the other enzyme must generate a four-nucleotide protruding 3' terminus. - [Read Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol]

Growing Bacteriophage M13 in Liquid Culture Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3994

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

Most manipulations with M13, including preparations of viral stocks and isolation of single- and double-stranded DNAs, begin with small-scale liquid cultures that are infected with an M13 plaque, picked from an agar plate. - [Read Growing Bacteriophage M13 in Liquid Culture Protocol]

Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3785

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how to identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with 32P-labeled double-stranded DNA rather than with antibodies. - [Read Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol]

Articles (1-3 of 3)

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.

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.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.

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