primers Protocols

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this protocol, double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: PCR Protocols

Protocol describes how double-stranded DNA probes, labeled in each strand, are produced in conventional PCRs containing equal concentrations of two primers, a double-stranded DNA template, three unlabeled dNTPs at concentrations exceeding the Km, and one [{alpha}-32P]dNTP at a concentration at or slightly above the Km (2-3 µm) for a thermostable DNA polymerase such as Taq. - [Read Radiolabeling of DNA Probes by the Polymerase Chain Reaction Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

Category: PCR Protocols: PCR Cloning Protocols

In this protocol sequences cloned in standard bacteriophage or plasmid vectors are amplified in PCRs containing primers targeted to flanking vector sequences.  The amplified fragments can be analyzed by gel electrophoresis, DNA sequencing, and/or restriction mapping.  Many colonies or plaques can be assayed simultaneously. - [Read Rapid Characterization of DNAs Cloned in Prokaryotic Vectors Protocol]

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

Long PCR Buffer, Cycle times and temperatures, Picking Primers, Hot Start Polymerases for Long PCR. Harvard-Lipper Center for Computational Genetics. Modified from Cheng et al. - [Read Reagents and Guidelines for Long PCR]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol uses FAM-(6-carboxy-fluorescein) or JOE-(6-carboxy-4', 5' -dichloro-2',7' -dimethoxy-fluorescein) labeled LUX (Light Upon eXtension) primers, which can quantify 100 or fewer copies of the target DNA in a background of nonspecific templates, over a broad dynamic range of less than 100-107 copies.  It uses uracil deglycosylase (UDG) to minimize the risk of carryover contamination, and includes a melting curve analysis of the product. - [Read Real-Time PCR Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In the first part of this protocol, the linear range of amplification is determined by carrying out 10 identical PCRs in the presence of [{alpha}-32P]dCTP and stopping one reaction after every two cycles.  Amplification products are quantified on a denaturing polyacrylamide gel and the results plotted on a graph (counts per minute vs. cycle number).  Total RNA is used as an internal control. - [Read Relative RT-PCR: Determining the Linear Range of Amplification and Optimizing the Primers:Competimer]

Category: Nucleic Acid Purification Protocols

In this protocol, ultrafiltration through Centricon or Microcon concentrators is used to remove unused primers, primer-dimers, and NTPs from preparations of amplified DNA. - [Read Removal of Oligonucleotides and Excess dNTPs from Amplified DNA by Ultrafiltration Protocol]

Category: PCR Protocols: PCR Cloning Protocols

Protocol exploits the discovery that Rnase A can efficiently cleave at single rC or rU bases embedded in double-stranded DNA.  Entire plasmid vectors are amplified using long, high-fidelity PCR with riboprimers, which carry a single rC residue at their 3' end.  Target DNA is amplified using similar primers, which also end in a rC residue. - [Read Ribocloning: DNA Cloning and Gene Construction Using PCR Primers Terminated with a Ribonucleotide]

Category: DNA Protocols: SNP Protocols

SNP detection with mutagenic primers. Input sequence will be searched to find changes in one nucleotide near the location of the SNP, so that mutagenic primers may be easily designed. Bikandi, J., San Millán, R., Rementeria, A., and Garaizar, J. In silico analysis of complete bacterial genomes: PCR, AFLP-PCR, and endonuclease restriction. Bioinformatics 2004 Mar 22;20(5):798-9. - [Read SNP detection with mutagenic primers]

Category: DNA Protocols: cDNA Library Protocols

Protocol describes the generation of cDNA probes from poly(A)+ mRNA using random oligonucleotide primers. Probes of this type are used for differential screening of cDNA libraries. - [Read Synthesis of cDNA Probes from mRNA Using Random Oligonucleotide Primers Protocol]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols

TAIL PCR Protocol. TAIL is a series of reactions that are intended to map where a T-DNA (transfer DNA) has inserted within the genome. The main components of the 3 reactions are the AD (Arbitrary Degenerate) primers, border primers, and DNA from the T-DNA - [Read TAIL PCR Protocol]

Category: PCR Protocols

TAIL PCR Protocol. TAIL is a series of reactions that are intended to map where a T-DNA (transfer DNA) has inserted within the genome. The main components of the 3 reactions are the AD (Arbitrary Degenerate) primers, border primers, and DNA from the T-DNA - [Read TAIL PCR Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

This protocol is designed to detect sequences in the murine genome by polymerase chain reaction amplification, and is adapted from Stratman and Simon (Transgenic Res. 12, 521-522 (2003)).For those familiar with PCR genotyping, this method differs from the typical protocol by utilizing a unique enzyme (Klentaq), 30mer primers, and a 68° annealing temperature. - [Read Universal Mouse Genotyping Protocol]

Category: Viral Protocols

Methods and information related to viral detection and concentration in food. Includes: Nucleic acid amplification methods; Recent progress in polymerase chain reaction detection; Detection of human enteric viruses in non-shellfish food; Choice of primers for the detection of human caliciviruses in foods; Biosensors and microarray detection; Alternative confirmation methods. - [Read Viral Detection and Concentration in Food]