polymerases Protocols

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: PCR Cloning Protocols

This method of direct cloning takes advantage of the unpaired adenosyl residue added to the 3' terminus of amplified DNAs by Taq and other thermostable polymerases. - [Read Cloning PCR Products into T Vectors 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: 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

With this protocol, transcripts that were initiated from specific genes by RNA polymerases prior to permeabilization can be measured. Instead of a nuclear extract, permeabilized cells are used. Includes information on: Permeabilization of Cells; In vitro Transcription Reaction (Run-off); Isolation of RNA; Preparation of Slot Blot Membrane for Hybridization; Hybridization of Nitrocellulose Membrane; TCA Precipitation to Determine Incorporation of [32P] GTP into Nucleic Acid - [Read In Vitro Transcription Assay (Run-off Assay) using Permeabilized Cells]

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: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes in vitro transcription with the SP6 polymerase.  For other polymerases (T3, T7), simply use the appropriate buffer and desired RNA polymerase instead of the SP6 polymerase. - [Read Preparation of dsRNA Molecules for RNAi in Mouse Oocytes and Early Embryos Protocol]

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

In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rRNA, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. - [Read Protocol: Purification of In Vitro Synthesized mRNA with Microcon or Centricon Centrifugal Filters]

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]

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]