primers Protocols

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "5'-end" partial cDNA clones using classic RACE, the first-strand products are generated by reverse transcription (primer extension) from a known gene-specific primer (GSP-RT).  Then, a poly(A) tail is appended using terminal deoxynucleotidyltransferase (Tdt) and dATP.  Amplification is carried out using three primers. - [Read 5'-End cDNA Amplification Using Classic RACE Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

Protocol for 96-well confirmation Yeast PCR. Includes: Clonal purification; Generate a master plate (96-well format); Making a frozen backup stock; Confirmation PCR for one Row; ORF Specific Confirmation PCR --> "A-B" primers (upstream junction); Transfer template DNA to multiwell PCR plate; Prepare and dispense master mix for A-B PCR. - [Read 96-Well Confirmation Yeast PCR Protocol]

Category: PCR Protocols

The MagneSil system can selectively isolate PCR products that are more than 150-bp long from primers and primer -dimers.  The technology can be used with a number of robotic workstations, including Beckman Coulter’s Biomek 2000 and FX Laboratory Automation Workstations.  The procedure can also be carried out manually.  Typical recovery is more than 80% for a 1-kb product with negligible carryover of primers or nucleotides. - [Read A Magnetic Particle-Based Method for Purifying PCR Products from Solution Protocol]

Category: PCR Protocols: AFLP PCR

Amplified Fragment Length Polymorphisms and Microsatellites: A phylogenetic perspective. Julian P. Robinson, Stephen A. Harris. What are AFLPs and how are they produced? How AFLPs have been used? Problems? Restriction Enzymes and Primers. AFLP Reproducib - [Read Amplified Fragment Length Polymorphisms and Microsatellites]

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: Colony PCR

Protocol describes the use of PCR to screen for bacteria that carry recombinant plasmids.  The PCR can be carried out using the same primers as for amplification of the cloned insert.  To determine the orientation of the insert, a third, insert-specific primer that is asymmetrically distanced from the clonal insertion site can be used. - [Read Colony PCR Protocol II]

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: DNA Protocols: DNA Sequencing Protocols

Protocol for cycle sequence reactions for large insert plasmid templates. Includes: CycleSequenceConditions; Primers Useful For BAC and PAC Vectors. - [Read Cycle Sequence Reactions For Large Insert Plasmid Templates Protocol]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Because sequencing reactions catalyzed by thermostable DNA polymerases such as Taq are carried out at elevated temperatures, problems caused by mismatched annealing of primers or templates rich in secondary structure are greatly alleviated. - [Read Dideoxy-mediated Sequencing of DNA Using Taq DNA Polymerase Protocol]

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

In this protocol asymmetric PCR is used to generate single-stranded DNA templates for dideoxy-sequencing - [Read Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers Protocol]

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: DNA Sequencing Protocols

Direct sequencing from amplified bacterial and large insert cloned human or mouse genomic DNA via an improved MultiPlex PCR-based method. Includes: Preparing primers for MP-PCR; Amplification; PCR Product Clean-up; Sequencing. - [Read Direct Sequencing Using MultiPlex PCR-Based Method]

Category: DNA Protocols: DNA Sequencing Protocols

Methods for DNA sequencing.  Includes: Bst-catalyzed radiolabeled DNA sequencing; Radiolabeled sequencing gel preparation, loading, and electrophoresis; Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye primers; Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye terminator reactions; Sequenase[TM] catalyzed sequencing with dye-labeled terminators; etc. - [Read DNA Sequencing Methods]

Category: PCR Protocols: PCR Optimization

Method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.  The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. - [Read Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization Protocol]

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Method assesses cellular mRNA transcripts in tissue sections and cell cultures using unique short anti-sense primers directed against sequences in particular protein(s). The unlabeled synthetic cDNA oligonucleotide primers are extended complementary to a sense mRNA transcript using reverse transcriptase and labeled through incorporation of a fluorescent-labeled dUTP nucleotide base. This procedure provides rapid detection of low abundance mRNA messages that can be related to other cellular.... - [Read Fluorescent In Situ Transcription in Cells and Tissues 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: 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: Plant Biology Protocols: Plant DNA RNA Isolation Protocols

Retroelements and their derivatives are a ubiquitous and abundant component of plant genomes. Major classes of retroelements include the Pseudoviridae (Ty1-copia ), the Metaviridae (Ty3 -gypsy) and the Retroposineae LINE (non-LTR) groups. All reverse transcribing elements can be included in a universal classification. Includes: Pseudoviridae (Ty1-copia) Degenerate Primers; Metaviridae (Ty3-gypsy) Element Degenerate Primers; LINE Element Degenerate Primers; PCR Programmes. - [Read Isolation of Retroelement from Plant Genomic DNA]

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: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

Information for oligonucleotide details required for PCR. Includes: Primer choice; UPTAG PRIMER; DNTAG PRIMER; UP_45 and DOWN_45 PRIMERS; UP_90 and DOWN_90 PRIMERS. - [Read Oligonucleotide Details for PCR]

Category: PCR Protocols: PCR Optimization

PCR primer design and pcr reaction optimization. Ed Rybicki. Factors Affecting the PCR, Denaturing Temperature and Time, Annealing Temperature and Primer Design, Primer Length, Degenerate Primers, Elongation , Temperature and Time, Reaction Buffer, Cycl - [Read PCR primer design and pcr reaction optimization.]

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: Laboratory Model Organisms Protocols

Genetic Protocols for Pristionchus pacificus. Includes: Freezing worms; EMS mutagenesis; Psoralen mutagenesis; Construction of deletion libraries to generate P. pacificus gene knock-outs; Designing primers for the gene of interest; RNAi and morpholino by injection. - [Read Pristionchus pacificus Genetic Protocols]

Category: RNAi Technology Protocols

Short interfering RNAs (siRNAs) can be used to prime RNA synthesis by the RNA-dependent RNA polymerase (RdRP).  SiRNAs can be used by RdRP as primers for specific cellular mRNAs, forming dsRNA products capable of inducing transitive RNAi. - [Read Protocol for siRNA-Primed RNA Synthesis Protocol]