templates Protocols

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol presents the amplification of insert end sequences from bacterial artificial chromosome clones using TAIL-PCR. The amplified products are suitable as probes for chromosome walking and genome mapping and as templates for direct sequencing. The protocol has been used in rice genome studies. - [Read Amplification of Insert End Sequences from BACs Clones by Thermal Asymmetric Interlaced PCR]

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

This protocol describes an in vitro transcription assay that allows for a single round of transcription from in vitro assembled chromatin. Comparing the activity of a receptor or transcriptional coactivator in an assay that measures only a single round of transcription with the results from multiple rounds of transcription can help elucidate the mechanism of transcriptional activation by those factors. - [Read Assay:Single Round of In Vitro Transcription from Assembled Chromatin Templates Using a HeLa Cell Ex]

Category: DNA Protocols: DNA Sequencing Protocols: Big Dye Protocols

Big Dye protocols and notes for: cosmid, BAC, BAC, Fosmid Templates. - [Read Big Dye Protocols and Notes - Cosmid, BAC, BAC, Fosmid Templates]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin 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

Protocol describes DNA-sequencing of single-stranded DNA templates in reactions catalyzed by Sequenase. - [Read Dideoxy-mediated Sequencing Reactions Using Bacteriophage T7 DNA Polymerase (Sequenase) 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: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol for dideoxy-mediated sequencing reactions using the Klenow fragment of E. coli DNA polymerase I and single-stranded DNA templates. - [Read Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase]

Category: E Coli Protocols: E coli Nucleic Acid Protocols

Protocol for dideoxy-mediated sequencing reactions using the Klenow Fragment of E. coli DNA polymerase I and single-stranded DNA templates. - [Read Dideoxy-mediated Sequencing Reactions Using the Klenow Fragment of E. coli DNA Polymerase I]

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: 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: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: Nucleic Acid Purification Protocols

Preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe.  At the end of the reaction nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by autoradiography or Southern hybridization.  Method used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. - [Read Mapping RNA with Nuclease S1 Protocol]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

Preparations of RNA containing an mRNA of interest are hybridized to a radiolabeled single-stranded RNA probe. The method can be used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. - [Read Mapping RNA with Ribonuclease and Radiolabeled RNA Probes Protocol]

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: DNA Protocols: DNA Sequencing Protocols: Big Dye Protocols

Protocol for plasmid templates for Big Dye. - [Read Plasmid Templates for Big Dye Protocol]

Category: DNA Protocols: DNA Sequencing Protocols

The inclusion of formamide in sequencing gels eliminates secondary structure in the DNA during electrophoresis. Formamide gels are particularly useful and almost a necessity when sequencing DNA templates with a G/C content >55%. - [Read Preparation of Denaturing Polyacrylamide Gels Containing Formamide Protocol]

Category: Nucleic Acid Electrophoresis Protocols

Protocol for preparation of denaturing polyacrylamide gels containing formamide.The inclusion of formamide in sequencing gels eliminates secondary structure in the DNA during electrophoresis.  Formamide gels are particularly useful and almost a necessity when sequencing DNA templates with a G/C content >55%. - [Read Preparation of Denaturing Polyacrylamide Gels Containing Formamide 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: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

Protocol describes how double-stranded plasmid DNA is denatured by alkali and annealed to an appropriate oligonucleotide primer, in preparation for dideoxy-sequencing reactions catalyzed by Sequenase. - [Read Preparing Denatured Templates for Sequencing by Dideoxy-mediated Chain Termination Protocol]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography. Fragments of DNA recovered from agarose gels are sometimes poor templates or substrates in subsequent enzymatic reactions.  This problem can be solved by binding the DNA to a positively charged matrix, such as DEAE-Sephadex or DEAE-Sephacel, in buffers of low ionic strength.  After washing the matrix, the DNA is eluted by raising the strength of the buffer. - [Read Purification of DNA Recovered Anion-exchange Chromatography Protocol]

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]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol is used to purify small amounts of bacteriophage {lambda} DNA that are suitable for use as substrates for restriction enzymes and templates for DNA and RNA polymerases. - [Read Rapid Analysis of Bacteriophage lambda Isolates: Purification of lambda DNA from Plate Lysates]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

This protocol is used to purify small amounts of recombinant DNAs cloned in robust strains of bacteriophage {lambda} such as {lambda}gt10, {lambda}gt11, {lambda}ZAP, or ZipLox. The DNAs are suitable for use as substrates for restriction enzymes and templates for DNA and RNA polymerases. - [Read Rapid Analysis of Bacteriophage {lambda} Isolates: Purification of {lambda} DNA from Liquid Cultures]