template Protocols

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: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol describes large fragment DNA replication by Adenovirus proteins using TP-DNA as template. - [Read Adenovirus DNA Replication Assay Protocol]

Category: Plant Biology Protocols: Plant Genetics Protocols

AFLP was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype. Protocol includes: Generate polymorphic recombinant F2 (or F3) population; Isolate genomic DNA; Restriction of DNA; Ligation of adapters; Pre-amplification of template DNA; AFLP-PCR; etc. - [Read AFLP For Positional Cloning]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR.  A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems.  The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization. - [Read Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

The ability to synthesize RNA in the lab is critical to many techniques.Radiolabeled and nonisotopically labeled RNA probes, generated in small scale transcription reactions can be used in blot hybridizations and nuclease protection assays. This article includes information on: Requirements For Transcription, RNA Phage Polymerases, Template Options: Plasmids, PCR Products, Oligonuclotides and cDNA, Sense or Antisense, Conventional Or Large Scale Synthesis, Products for In Vitro Transcription. - [Read Basic Information on In Vitro Transcription]

Category: PCR Protocols: cDNA Synthesis Protocols

This method, for the selective amplification of full-length cDNA ends, involves the addition of an adapter during reverse transcription.  This method takes advantage of the propensity of Moloney murine leukemia virus reverse transcriptase (MMLV RT) to append two to four cytosines to the 3'-end of newly synthesized cDNA strands.  The additional residues are added when the enzyme reaches the 5'-cap structure at the end of the mRNA template. - [Read Cap-Switching RACE Protocol]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol described here produces chromatin with regularly spaced nucleosomes having physiological nucleosome repeat lengths; something that can be difficult to achieve with purified components. In addition, chromatin assembled with the S-190 Chromatin Assembly Extract contains the ATP-dependent chromatin remodeling factors necessary for efficient transcription. - [Read Chromatin Assembly on Template DNA with Transcription Factors and Drosophila S-190 Chromatin Assembl]

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: BACs Bacterial Artificial Chromosome Protocols

Sequencing conditions tested for the ABI Big-Dye terminators (PE-ABI #4303150 for the 1000 reaction kit - Description: TF,KIT BTD RR-1000) with various templates. Important to quantitate all templates by agarose gel electrophoresis vs size and concentration standards and do a few tests with different template concentrations to determine the optimal conditions for your reactions. Several conditions are given. - [Read Dye Protocols and Notes for Cosmid, BAC, BAC, Fosmid Templates]

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: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Provides information on specific parameters you need to be aware of when using eukaryotic in vitro translation systems. Includes: DNA Template Considerations; Protein Labeling; Non-Radioactive Protein Labeling. - [Read Eukaryotic In Vitro Translation Systems]

Category: DNA Microarray Protocols

This protocol describes the steps required to produce a cDNA microarray. Gene-specific DNA is produced by PCR amplification of purified template plasmid DNAs from cloned ESTs. The PCR product is purified by ethanol precipitation, thoroughly resuspended in - [Read Fabrication Protocol for DNA Microarrays]

Category: DNA Protocols: SNP Protocols

FP-TDI Method for SNP Detection. The FP-TDI protocol was originally reported by Drs. Chen, Levine, and Kwok at Washington University in 19991,2. FP-TDI stands for template directed dye terminator incorporation assay with detection by fluorescence polarization. It is a single base primer extension assay couple with homogeneous FP detection. Perkin Elmer - [Read FP-TDI Method for SNP Detection]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for in vitro mutagenesis using double-stranded DNA templates. Two oligonucleotides are used to prime DNA synthesis catalyzed by a high-fidelity thermostable polymerase on a denatured plasmid template. The two oligonucleotides both contain the desired mutation and occupy the same starting and ending positions on opposite strands of the plasmid DNA. - [Read In Vitro Mutagenesis Using Double-stranded DNA Templates: Selection of Mutants with DpnI]

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

Protocol illustrates the rules of successful long PCR: No more than 1 ng of template DNA is used per microliter of PCR in a 100-µl reaction; approximately 0.1 µl of KlentaqLA (not plain Taq) is used per kilobase of target (for targets >10 kb, 1-1.3 µl of enzyme should be used); the Mg++ concentration is considered as the excess over the level of dNTPs. - [Read Long and Accurate PCR Protocol]

Category: PCR Protocols: Long PCR Protocol. Extended PCR.

Long Template PCR. Troubleshooting Long PCR, Amplification of Long PCR Fragments, Roche Guide. - [Read Long Template PCR. Roche Guide.]

Category: DNA Microarray Protocols

This protocol describes clone handling, plate replication, and DNA template preparation in a 96 well format. Hasseman. TIGR Microarray Protocols - [Read MICROARRAY cDNA CLONE GROWTH AND TEMPLATE]

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: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol describes a system which includes all of the necessary components for in vitro transcription as well as a positive control template that provides run-off transcripts from a CMV immediate early promoter. This system is designed for runoff transcription. Alternatively, transcription products can be analyzed by primer extension. - [Read Nuclear Extract in vitro Transcription System]

Category: Mouse Protocols: Mouse Genetics Protocols

Method is used chiefly to genotype transgenic and knockout mice. Each 6-10-mm snippet of mouse tail yields 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and as a template in PCRs. - [Read Preparation of Genomic DNA from Mouse Tails and Other Small Samples Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

Simple protocol is used to extract DNA from small numbers of cultured cells and from fragments of soft or bony tissues.  The method is used chiefly to genotype transgenic and knockout mice.  Each 6-10-mm snippet of mouse tail yields 50-100 µg of DNA that can be used in dot or slot blotting to detect a transgene of interest, in Southern hybridization to detect DNA fragments that are <20 kb in size, and as a template in PCRs. - [Read Preparation of Genomic DNA from Mouse Tails and Other Small Samples Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Single-stranded templates of bacteriophage M13 DNA containing 20-30 residues of uracil in place of thymine are generated during growth of the bacteriophage in an F' strain of E. coli carrying mutations in the ung and dut genes. This DNA is used as a template in the Kunkel method of oligonucleotide-directed mutagenesis (Oligonucleotide-directed Mutagenesis of Single-stranded DNA). - [Read Preparation of Uracil-containing Single-stranded Bacteriophage M13 DNA Protocol]

Category: RNA Protocols: RT-PCR and cDNA synthesis

Protocol for First-strand cDNA Synthesis (PCR template generation with M-MuLV Reverse Transcriptase) - [Read Protocol for First-strand cDNA Synthesis Fermentas]

Category: PCR Protocols: Quantitative PCR Protocols

Quantitative PCR involves co-amplification of two templates: a constant amount of a preparation containing the desired target sequence and serial dilutions of a reference template that is added in known amounts to a series of amplification reactions.  The concentration of the target sequence is determined by simple interpolation into a standard curve. - [Read Quantitative PCR Protocol II]