termini Protocols

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: 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: 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: 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: 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: 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]

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: 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]

Category: PCR Protocols

Method describes how to modify the termini of PCR products by introducing restriction sites and other features.  To reduce the chance of contamination with exogenous DNAs, prepare and use a special set of reagents and solutions for PCR only.  Bake all glassware for 6 hours at 150°C and autoclave all plasticware. - [Read Genetic Engineering with PCR Protocol]

Category: PCR Protocols: PCR Optimization

Method describes how to modify the termini of PCR products by introducing restriction sites and other features.  To reduce the chance of contamination with exogenous DNAs, prepare and use a special set of reagents and solutions for PCR only.  Bake all glassware for 6 hours at 150°C and autoclave all plasticware. - [Read Genetic Engineering with PCR Protocol]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for phosphorylating the 5' termini of oligonucleotides. - [Read Phosphorylating the 5' Termini of Oligonucleotides Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

3'-RACE reactions are used to isolate unknown 3' sequences or to map the 3' termini of mRNAs onto a gene sequence.  3'-RACE requires knowledge of a small region of sequence within either the target RNA or a partial clone of cDNA.  A population of mRNAs is transcribed into cDNA with an adaptor-primer consisting at its 3' end of a poly(T) tract and at its 5' end of an arbitrary sequence of 30-40 nucleotides. - [Read Rapid Amplification of 3' cDNA Ends 3'-RACE Protocol]

Category: DNA Protocols: T4 Polynucleotide Kinase Protocols

T4 Polynucleotide Kinase Protocol Promega. 5´ End-Labeling Protocol, Non-Radioactive Kinase Reaction, Exchange Reaction Protocol Using Protruding 5´ Phosphoryl Termini as Templates. Promega. T4 PNK Protocols. - [Read T4 Polynucleotide Kinase Protocol Promega]