cloning Protocols

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

Mannie Liscum and Paul Oeller. Department of Plant Biology. Carnegie Institution of Washington, Stanford. AFLP technology is used here to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been i - [Read AFLP: not only for fingerprinting, but for positional cloning]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Removal of the 5'-phosphate groups from the internal termini of bacteriophage {lambda} arms is used to prevent self-ligation and suppress the background of nonrecombinant bacteriophages during cloning. - [Read Alkaline Phosphatase Treatment of Bacteriophage lambda Vector DNA Protocol]

Category: DNA Protocols: DNA Extraction Protocols: Extracting Ancient DNA

The DNA obtained was invariably of a low average molecular size and damaged by oxidative processes, but PCR can be used to amplify and study short mitochondrial DNA sequences thatare of anthropological and evolutionary significance. SVANTE PAABO, PNAS. - [Read Ancient DNA: Extraction, characterization, molecular cloning, and enzymatic amplification. PDF]

Category: RNAi Technology Protocols

Protocol describes the preparation of an shRNA-expressing Gateway entry vector.  This process is preceded by the design and synthesis of target gene-specific sense and antisense oligonucleotides which, when annealed, will constitute an shRNA cassette. - [Read Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

This assay is used when working with phage vectors carrying the beta-gal gene. If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-gal gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta-gal will work as indicator host bacteria; a single chromosomal copy of the gene is not a problem. - [Read Assay for Phage Containing the Beta-galactosidase Gene]

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

This assay is used when working with phage vectors carrying the beta-galactosidase gene (often used for immunological screening). If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-galactosidase gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta- galactosidase will work as indicator host bacteria. - [Read Assay for Phage Containing the Beta-galactosidase Gene Protocol]

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]

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: 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: Cloning Protocols: Vector Protocols

Protocol for blunt-ended cloning into plasmid vectors. - [Read Blunt-ended Cloning into Plasmid Vectors Protocol]

Category: RNA Protocols: cDNA Libraries Library

Protocol for cDNA synthesis and cloning cDNA into plasmid vector. 1st Strand cDNA Synthesis, and determine the efficiency of first strand cDNA synthesis. Also includes second Strand cDNA Synthesis. Dr.Frank - [Read cDNA Synthesis and Cloning]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Chromatin Immunoprecipitation (ChIPs) Cloning Protocol Farnham Lab - [Read Chromatin Immunoprecipitation (ChIPs) Cloning Protocol]

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

Protocol for cloning genes from a phage library. Includes: Titer and plate out phage; Lift plaques onto filters and prepare them for screening; Make a probe; Hybridize the probe to the filters; Wash the filters and expose to film; Purify putative plaques; Excise plasmid from the desired phage. - [Read Clone Genes From a Phage Library Protocol]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Cloning Enzymes a Guide Promega. An Enzyme Resource Guide series, highlights those enzymes important in nucleic acid cloning procedures. Promega - [Read Cloning Enzymes a Guide Promega]

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: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol for cloning of amplified miRNA target sites into 3'-UTR of luciferase reporter vector. - [Read Cloning of Amplified miRNA Target Sites into 3'-UTR of Luciferase Reporter Vector 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: PCR Protocols: PCR Cloning Protocols

This method of direct cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable polymerases. - [Read Cloning PCR Products into T Vectors 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: Cloning Protocols: Vector Protocols

Protocol for cloning siRNA duplexes into pSUPER or pSUPER retro vectors. - [Read Cloning siRNA Duplexes into pSUPER or pSUPER Retro Vectors Protocol]

Category: RNAi Technology Protocols

Protocol for cloning siRNA duplexes into pSUPER or pSUPERretro vectors. - [Read Cloning siRNA Duplexes into pSUPER or pSUPERretro Vectors 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]