amplify Protocols

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: Cell Culture Protocols: Insect Cell Culture Protocols

The use of Ferbach flasks is an ideal method to amplify the cells and virus inoculum for the bioreactor. The method is also very practical for producing sufficient amounts of various proteins for feasibility studies using HyQ© SFX-Insect™ medium. The large surface area in this vessel enables sufficient aeration and, therefore, the cells, when they reach high density, are not oxygen depleted. - [Read Culturing of Insect Cells and Production of Baculovirus Expressed Recombinant Proteins in 2.8 Liter]

Category: PCR Protocols

Method uses PCR to amplify and display many cDNAs derived from the mRNAs of a given cell or tissue type.  The method relies on two different types of synthetic oligonucleotides: anchored antisense primers and arbitrary sense primers.  A typical anchored primer is complementary to approx. 13 nucleotides of the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. - [Read Differential Display-PCR Protocol]

Category: DNA Protocols: Genomic DNA Library Protocols

Protocol describes how to construct and amplify a genomic DNA library in a plasmid vector (pSPL3) equipped to express cloned exons in transfected mammalian cells. - [Read Exon Trapping and Amplification for Construction of the Library Protocol]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Protein Interactions Protocols

This protocol describes the first step in constructing an array: amplification of the predicted ORFs that are to be included in the array. Gene-specific primers containing vector-specific flanking sequences that facilitate recombinational cloning are used to amplify each ORF. A secondary amplification can be used to extend the length of the homologous vector sequence flanking the ORF. - [Read Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Amplification of ORFs]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to amplify DNA from macroconidia. - [Read How to Amplify DNA from Macroconidia]

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]

Category: PCR Protocols: PCR Cloning Protocols

Protocol describes the use of vectorette PCR and single-site PCR to amplify the terminal sequences of genomic sequences cloned in high-capacity vectors such as PACs and YACs. - [Read Isolating the Ends of Genomic DNA Fragments Cloned in High-capacity Vectors]

Category: PCR Protocols: Standard PCR Protocol

Protocol can be used to amplify DNA up to 25 kb in length.  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 Long PCR Protocol]

Category: PCR Protocols: cDNA Synthesis Protocols

In MOPAC, the amino-terminal and carboxy-terminal sequences of a peptide are used to design two redundant families of oligonucleotides encoding the aminoand carboxy-terminal sequences of the peptide.  The primers are used either to amplify a segment of cDNA prepared by RT-PCR from a tissue known to express the protein or to amplify a segment of DNA from an established genomic or cDNA library. - [Read Mixed Oligonucleotide-primed Amplification of cDNA MOPAC Protocol]

Category: PCR Protocols: Real-Time PCR Protocols

In multiplex real-time PCR, different sets of primers with different labels are used to amplify separate genes from the template DNA in one tube.  This protocol uses LUX (Light Upon eXtension) primers from invitrogen.  FAM (6-carboxy-fluorescein) is used to label the gene of interest, and JOE (6-carboxy-4', 5'-dichloro-2',7'-dimethoxy-fluorescein) is used to label a housekeeping gene as an internal control to normalize between different reactions. - [Read Multiplex Real-Time PCR Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Primer pairs will amplify sequences present as a single copy in the mouse genome with the Universal Genotyping Protocol. Includes: b-Galactosidase (LacZ); cre-recombinase; CFP; diphtheria toxin; dsRED; Fabpi-200; Fabpi-500; flp recombinase; GFP/BFP/YFP; human growth hormone (complete); human growth hormone (transcriptional stop); luciferase (click-beetle); luciferase (firefly); neomycin phosphotransferase; SRY (male-specific); tTA (tet-on). - [Read PCR Genotyping Primer Pairs Protocols]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: PCR Optimization Protocols

The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. P - [Read PCR Program Design]

Category: PCR Protocols: PCR Optimization

The requirement of an optimal PCR reaction is to amplify a specific locus without any unspecific by-products. Therefore, annealing needs to take place at a sufficiently high temperature to allow only the perfect DNA-DNA matches to occur in the reaction. P - [Read PCR Program Design]

Category: PCR Protocols

DNA prepared by PCR-mediated gene disruption can be used to transform yeast in gene replacement experiments.  This protocol uses two primers, tailed with approximately 50 nucleotides homologous to the gene of interest, that target insertion of the PCR product to that locus.  Each primer ends with a universal sequence that is designed to amplify various selectable markers from plasmid templates. - [Read PCR-Mediated Gene Disruption: One-Step Method Protocol]

Category: PCR Protocols: Standard PCR Protocol

Protocol describes how to amplify a segment of double-stranded DNA in a chain reaction catalyzed by a thermostable DNA polymerase.  It is the foundation for all subsequent variations of the polymerase chain reaction. - [Read The Basic Polymerase Chain Reaction Protocol]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for transposon mediated mutagenesis. Includes: Amplify ORF from MG1655; Transposition reaction; Transformation; Picking; Verify mutation by Culture PCR; Confirm mutations by sequencing; Curing the temperature sensitive pKD46 plasmid. - [Read Transposon Mediated Mutagenesis Protocol]

Category: Bacteria Genetics Protocols

Protocol for transposon-mediated mutagenesis. Includes: Amplify ORF from MG1655; Transposition reaction; Transformation; Picking; Verify mutation by Culture PCR; Confirm mutations by sequencing; Curing the temperature sensitive pKD46 plasmid. - [Read Transposon-Mediated Mutagenesis Protocol]