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A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol - http://www.aspergillus.org.uk/indexhome.htm?secure/laboratory_protocols/./genedel.html~main

Category: Fungi Protocols: Aspergillus Protocols

The technique makes use of an Escherichia coli strain expressing the redΑßΓ operon under the control of an inducible promoter. This enables the strain to carry out homologous recombination with only 50-60 bp of homologous sequence. The procedure does not require any DNA ligation and is very rapid. It allows a single gene or region on a cosmid to be replaced by a bi-functional selectable marker (having both an E. coli and an A. fumigatus marker). - [Read A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol]

Addition of linkers/adaptors to blunt-end DNA fragments PDF - http://www.oup.co.uk/pdf/pas/9v1-7-11.pdf

Category: Molecular Biology Protocols: DNA Ligation Protocols

Addition of linkers/adaptors to blunt-end DNA fragments. Ligation of mixture containing DNA and linkers/adaptors. Powell Gannon. Oxford Practical Approach Series. - [Read Addition of linkers/adaptors to blunt-end DNA fragments PDF]

AFLP For Positional Cloning - http://www-ciwdpb.stanford.edu/publications/methods/aflp.html

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]

AFLP PCR Protocol PDF - http://www4.ncsu.edu/~wagebrey/AFLP-Salm.pdf

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]

AFLP protocol - http://www.msu.edu/user/hazensam/aflp/AFLPprotocolMSU.html

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]

AFLP protocol Wolf Lab - http://bioweb.usu.edu/wolf/aflp_protocol.htm

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]

Alkaline Phosphatase Treatment of Bacteriophage lambda Vector DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3976

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]

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]

Bidirectional Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4139

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]

Blunt-end Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3830

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]

C. elegans RNAi Protocol - http://www.zoology.ubc.ca/~alorch/rnai-protocol.htm

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]

Cloning into Bacteriophage M13 Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4018

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]

Cloning protocols/tips by Astrid PDF - http://www.its.caltech.edu/~bjorker/Protocols/cloning%20protocols%20by%20Astrid.pdf

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]

Cohesive End Ligation - http://www.biochem.ucl.ac.uk/bsm/nmr/protocols/protocols/ligation.html

Category: Molecular Biology Protocols: DNA Ligation Protocols

DNA ligation protocol with excellent notes and tips. Chen Lab, Univ. College London. - [Read Cohesive End Ligation]

Construction of Genomic DNA Libraries in Cosmid Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4000

Category: DNA Protocols: Cosmid Library Protocols

Protocol describes how to construct a library of 35-45-kb fragments of genomic DNA in the double cos site cosmid vector, SuperCos-1. The steps include: Linearization and dephosphorylation of SuperCos-1 DNA; Partial digestion of high-molecular-weight DNA with MboI; Dephosphorylation of high-molecular-weight genomic DNA; Ligation of cosmid arms to genomic DNA: Packaging and plating recombinants; Isolation and analysis of recombinant cosmids: Validation of the library. - [Read Construction of Genomic DNA Libraries in Cosmid Vectors Protocol]

Direct Retrieval of DNA Fragments from Pulsed-field Gels Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4083

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]

Directional Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4140

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]

DNA cloning Procedures - http://hulab.tamu.edu/Protocols/cloning/

Category: DNA Protocols: DNA Cloning

Agarose gel purification, Annealing and extending, oligonucleotides, Ethanol Precipitation, Ligation Miniprep, Oligonucleotide purification, Recovering DNA bands, Restriction digest Gene Clean. The Hu Lab. - [Read DNA cloning Procedures]

DNA Insert Ligation into Vector DNA (with T4 DNA Ligase) - http://www.fermentas.com/techinfo/modifyingenzymes/protocols/p_dnainslig.htm

Category: Molecular Biology Protocols: DNA Ligation Protocols

Using digested vector DNA (50-400ng) and foreign DNA to be inserted. Fermentas - [Read DNA Insert Ligation into Vector DNA (with T4 DNA Ligase)]

DNA Ligation - http://www.bio.com/protocolstools/protocol.jhtml?id=p2053

Category: Molecular Biology Protocols: DNA Ligation Protocols

Description and protocol of the steps required to join together plasmid and insert fragments to create a new plasmid. Paul Kaufman, Univ California. - [Read DNA Ligation]

DNA Ligation Protocol from Takara Shuzo Co. - http://www.hawaii.edu/microbiology/MO/ligation.htm

Category: Molecular Biology Protocols: DNA Ligation Protocols

Insertion of DNA into Plasmid Vectors, Insertion of DNA into l Phage Vectors, Self-Circularization of Linear DNA (Intramolecular Ligation), Linker (Adaptor) Ligation. Alam's Lab Univ Hawaii. - [Read DNA Ligation Protocol from Takara Shuzo Co.]

DNA Ligation Protocol PDF - http://www.oup.co.uk/pdf/pas/9v1-7-9.pdf

Category: Molecular Biology Protocols: DNA Ligation Protocols

DNA ligation Method, Equipment and reagents, T4 DNA ligase. Gannon and Powell. Oxford Practical Approach Series. - [Read DNA Ligation Protocol PDF]

DNA ligation with Linearized DNA - http://www.genome.ou.edu/protocol_book/protocol_partII.html#II.D

Category: Molecular Biology Protocols: DNA Ligation Protocols

DNA ligations are performed by incubating DNA insert with linearized cloning vector in the presence of ligation buffer, rATP, and T4 DNA ligase. Roe. Univ. Oklahoma. - [Read DNA ligation with Linearized DNA]

EMBL Cloning protocols - http://www.embl-hamburg.de/display?file=~geerlof/webPP/protocoldb/Cloning/pdb_cloning.html

Category: DNA Protocols: DNA Cloning

Preparation of oligo solutions PCR experiments, Digestion of insert DNA, Digestion and dephosphorylation of vector DNA, Ligation of DNA fragments with sticky ends, Ligation of DNA fragments with blunt ends, Preparation of chemically competent E. coli - [Read EMBL Cloning protocols]

Inverse PCR Protocol II - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3487

Category: PCR Protocols: Inverse PCR Protocols

Inverse PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available. The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in the PCR. - [Read Inverse PCR Protocol II]

Articles (1-2 of 2)

Populational Analysis of Genomic DNA Protocol

A protocol on the populational Analysis of genomic DNA.

DNA Ligation Protocol

The DNA Ligation protocol described here contains the steps required to join together using ligase enzyme both plasmid DNA and insert DNA fragments in order to create a new plasmid. This new ligated plasmid can be transformed after into competent bacteria to produce DNA for mini, midi or maxi-prep isolation.

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