restriction Protocols

Category: Epigenetics and Methylation Protocols

The pattern and extent of DNA methylation can significantly affect the success of restriction digestions or bacterial transformations. Prokaryotic DNA may be methylated by host restriction/modification systems, while eukaryotic DNA often is methylated i - [Read A practical guide to DNA methylation Promega PDF]

Category: Plant Biology Protocols: Plant Genetics Protocols

AFLP was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype. Protocol includes: Generate polymorphic recombinant F2 (or F3) population; Isolate genomic DNA; Restriction of DNA; Ligation of adapters; Pre-amplification of template DNA; AFLP-PCR; etc. - [Read AFLP For Positional Cloning]

Category: PCR Protocols: AFLP PCR

AFLP Procecure, includes restriction Digestion of Genomic DNA. Michael Havey - Vegetable Genetics Laboratory - [Read AFLP Method]

Category: PCR Protocols: AFLP PCR

Restriction digestion, Adapter ligation, Preamplification, Final Amplification steps for AFLP PCR. Detailed protocol. Wondwossen Abebe Gebreyes - [Read AFLP PCR Protocol PDF]

Category: PCR Protocols: AFLP PCR

Restriction Digestion of Genomic DNA , Adapter preparation, Ligation of Adapters. Pre amplification Reactions. Gel electrophoresis, Silver Staining. AFLP Protocol and procedure. - [Read AFLP protocol]

Category: PCR Protocols: AFLP PCR

Restriction and Ligation reactions, Setting up the reactions, AFLP PCR reactions protocols. Analyzing the data using Genescan. Paul G. Wolf, Utah State Univ. - [Read AFLP protocol Wolf Lab]

Category: PCR Protocols: AFLP PCR

P Vos, R Hogers, M Bleeker, M Reijans, T van de Lee, M Hornes, A Frijters, J Pot, J Peleman, and M Kuiper. 1995. Nucleic Acid Research. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic - [Read AFLP: a new technique for DNA fingerprinting.]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Alphabetic List of Commercial Isoschizomers and Corresponding Fermentas Restriction Endonucleases. Fermentas - [Read Alphabetic List of Commercial Isoschizomers and Corresponding Fermentas Restriction Endonucleases]

Category: PCR Protocols: AFLP PCR

Amplified Fragment Length Polymorphisms and Microsatellites: A phylogenetic perspective. Julian P. Robinson, Stephen A. Harris. What are AFLPs and how are they produced? How AFLPs have been used? Problems? Restriction Enzymes and Primers. AFLP Reproducib - [Read Amplified Fragment Length Polymorphisms and Microsatellites]

Category: PCR Protocols: AFLP PCR

An introduction to AFLP and fAFLP. Mark E. Berres, University of Wisconsin. Amplified fragment-length polymorphism (AFLP) or its fluorescent version (fAFLP) is a PCR-based fingerprinting technology. AFLP basically involves the restriction of genomic DNA - [Read An introduction to AFLP and fAFLP]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Very detailed class protocol for Analysis of DNA by Restriction Enzyme Cleavage and Agarose Gel Electrophoresis. David Ng, UBC. - [Read Analysis of DNA by Restriction Enzyme Cleavage and Agarose Gel Electrophoresis]

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]

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for bidirectional, blunt-end cloning of DNA fragments.  The target DNA is PCR amplified and 3'-extensions are polished with Pfu DNA polymerase.  The amplicon is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction are used to transform competent Escherichia coli.  A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Bidirectional Cloning of PCR Products Protocol]

Category: Epigenetics and Methylation Protocols

Bisulfite-PCR for Restriction Analysis and/or Sequencing. Protocol written by Jean-Pierre Issa. M.D Anderson Cancer Centre Texas. - [Read Bisulfite-PCR for Restriction Analysis and/or Sequencing]

Category: PCR Protocols: Bisulfite Conversion Based PCR Protocols

Protocol for bisulfite-PCR for restriction analysis and/or sequencing. Bisulfite-PCR followed by restriction is a rapid and semi-quantitative method of analyzing DNA methylation.  The PCR products are also suitable for either direct sequencing or cloning and sequencing.  The most important step here is primer selection. - [Read Bisulfite-PCR for Restriction Analysis and/or Sequencing Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol for blunt-end cloning of PCR products. Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids.  The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves.  I - [Read Blunt-end Cloning of PCR Products Protocol]

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

Protocol for C. elegans RNAi. Includes: Transformation of competent cells; Blunt-end ligation; Preparation of competent cells; Dephosphorylation of linear plasmid DNA; Restriction Digest: EcoRV; Insert amplification from gDNA; Gel purification: QiaQuick gel purification kit; Mini-prep; Transformant Screening; Bacterial preparation and induction; Preparation of worms for RNAi feeding. - [Read C. elegans RNAi Protocol]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Cleavage Efficiency Close to the Termini of PCR Fragments. List of restriction enzymes and their cleavage efficiencies near the ends of DNA. Fermentas - [Read Cleavage Efficiency Close to the Termini of PCR Fragments]

Category: Genetics and Genomics: Cancer Genetics Protocols

Investigators can utilize X chromosome inactivation (methylation) to determine the clonality status of a tumor or premalignant lesion in females. The technique is based on a methylation-sensitive restriction enzyme and analysis of a polymorphic locus on the X chromosome. Clonal cell populations will show "loss" of the non-methylated allele after restriction digest. The assay can be performed on DNA recovered from microdissected samples. Both frozen tissue and fixed-embedded tissue can be used. - [Read Clonality - X Chromosome Inactivation Assay Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol describes three standard methods to construct bacteriophage M13 recombinants: (1) ligating insert DNA to a linearized vector, prepared by cleavage of M13 RF with a single restriction enzyme; (2) using alkaline phosphatase to suppress self-ligation of the linearized vector, and (3) using M13 RF cleaved with two restriction enzymes for directional cloning. - [Read Cloning into Bacteriophage M13 Vectors Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Pairs of oligonucleotide primers used in PCR are often designed with restriction sites in their 5' regions.  In many cases, the sites are different in the two primers.  In this case, amplification generates a target fragment whose termini now carry new restriction sites that can be used for directional cloning into plasmid vectors.  The purified fragment and the vector are digested with the appropriate restriction enzymes, ligated together, and transformed into E. coli. - [Read Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol]

Category: DNA Protocols: DNA Cloning

Cloning protocols/tips by Astrid. PCR and primer design, Extracting DNA from gel, Restriction of vector, Ligation, and Transformation. CalTech - [Read Cloning protocols/tips by Astrid PDF]

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

Protocol for direct retrieval of DNA fragments from pulsed-field gels. A gel slice containing a fragment of DNA resolved by pulsed-field gel electrophoresis is treated with agarase.  The released DNA can be used as a substrate for ligation or restriction without further purification. - [Read Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol]

Category: Cloning Protocols: Vector Protocols

Directional cloning requires that the plasmid vector be cleaved with two restriction enzymes that generate incompatible termini and that the fragment of DNA to be cloned carries termini that are compatible with those of the doubly cleaved vector. - [Read Directional Cloning into Plasmid Vectors Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol is for directional blunt-end cloning of DNA fragments.  The target DNA is PCR-amplified, 3'-extensions are polished with Pfu DNA polymerase, and the amplicon is ligated to a blunt-ended plasmid DNA.  The products of the ligation reaction are used to transform competent Escherichia coli.  A restriction enzyme is added to the ligation reaction to relinearize any self-religating vector DNA. - [Read Directional Cloning of PCR Products Protocol]