blunt Protocols

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]

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: 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: 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: 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]

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]

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]

Category: DNA Protocols: TOPO Cloning

Zero Blunt TOPO PCR Cloning Kit, Detailed protocol. (Invitrogen Life technologies). Alam Lab. Department of Microbiology, University of Hawai'i. - [Read Zero Blunt TOPO PCR Cloning Kit Protocol]