Water

Betsy Boedeker,* Gregory A. Wemhoff, Andrea Spencer, Roger Chiu, Suzanne Miller, Henry George, and Edward J. Weinstein
Betsy Boedeker, Gregory A. Wemhoff, Andrea Spencer,Roger Chiu, Suzanne Miller, Henry George and Edward J. Weinstein
LSI Volume 7 Article 1
Keywords: Amplification, Antibiotics, Cellular processes, Drug discovery, Functional genomics, Gene expression, Genomics, Molecular biology, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Reductions, Sequences, Transcription, Transduction, Vectors

*Calculations provide estimated results based on Antoine's Coefficients and their associated temperature range values. Antoine's Coefficients were obtained from the National Institute of Standards an...

Note: JumpStart Taq DNA polymerase has been shown to work effectively with up to 5% v/v DMSO. Other co-solvents, solutes (e.g., salts) and extremes in pH or other reaction conditions may reduce the a...
Keywords: AGE, Amplification, Electrophoresis, Evaporation, Gel electrophoresis, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Reductions, Size-exclusion chromatography, Solvents, Titrations

Note: The use of DMSO or formamide with JumpStart REDTaq DNA Polymerase is not recommended due to interference with the enzyme-antibody complex. Other co-solvents, solutes (e.g., salts) and extremes ...
Keywords: Amplification, Evaporation, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Reductions, Size-exclusion chromatography, Solvents, Titrations

Note: JumpStart Taq DNA polymerase has been shown to work effectively with up to 5% v/v DMSO. Other co-solvents, solutes (e.g., salts) and extremes in pH or other reaction conditions may reduce the a...
Keywords: AGE, Amplification, Electrophoresis, Evaporation, Gel electrophoresis, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Reductions, Size-exclusion chromatography, Solvents, Titrations

Restorase® DNA Polymerase with 10x Reaction Buffer combines Sigma's Long and Accurate enzyme technology with a DNA repair enzyme resulting in a blend that facilitates repair and amplification of dama...
Keywords: AGE, Alkylations, Amplification, Buffers, Catalysis, Degradations, Diagnostic, Electrophoresis, Gas chromatography, Gel electrophoresis, Melting, Nucleic acid annealing, Nucleic acid denaturation, Nucleic acid hybridization, Polymerase chain reaction, Precipitation, Size-exclusion chromatography

REDTaq DNA Polymerase is a convenient package that includes all the necessary components for a PCR reaction except primers, DNA template and water. The formulation has been optimized for amplificatio...
Keywords: AGE, Amplification, Buffers, Digestions, Electrophoresis, Evaporation, Gel electrophoresis, Polymerase chain reaction, Purification, Sequencing, transformation

During PCR assay preparation, nonspecific amplification can occur due to binding of PCR primers to nonspecific templates and from formation of primer dimers which result from using other primer molec...
Keywords: AGE, Amplification, Buffers, Electrophoresis, Enzyme activity, Gel electrophoresis, Nucleic acid denaturation, Polymerase chain reaction, Size-exclusion chromatography

Technology Overview Equipment Reagents Assay Considerations Procedure Troubleshooting Materials References
Keywords: Amplification, Buffers, Cloning, Digestions, Electrophoresis, Evaporation, Gas chromatography, Gel electrophoresis, Gene expression, Melting, Nucleic acid annealing, Nucleic acid denaturation, Peptide synthesis, Phase transitions, Polymerase chain reaction, Purification, Reductions, Sequencing, Size-exclusion chromatography, transformation

One approach to assay optimization is to determine the optimum annealing temperature (Ta) of the primers by testing identical reactions containing a fixed primer concentration, across a range of anne...
Keywords: Amplification, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography

The single most important step in assuring success with PCR is high quality DNA preparation. Integrity and purity of DNA template is essential. Quantitative PCR involves multiple rounds of enzymatic ...
Keywords: AGE, Amplification, Diagnostic, Electrophoresis, Gas chromatography, Gel electrophoresis, Gene expression, Melting, Molecular probes, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Separation, Size-exclusion chromatography, Solvents

· How do Hot-Start dNTPs work? · Handling · Protocols for Taq DNA Polymerase—Standard PCR, Fast PCR, Multiplexed PCR and Real-Time PCR · Troubleshooting · Standard Thermal Cycling Conditions for Othe...
Keywords: AGE, Amplification, Buffers, Centrifugation, Degradations, Electrophoresis, Gel electrophoresis, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Purification, Size-exclusion chromatography

KiCqStart SYBR Green qPCR ReadyMix is a 2X concentrated, ready-to-use reaction cocktail that contains all components, except primers and template, for real-time quantitative PCR (qPCR). This unique c...
Keywords: Amplification, Gene expression, Molecular probes, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography, Titrations

Effective denaturation is accomplished by the use of higher temperatures for shorter periods of time or by the use of co-solvents, such as dimethyl sulfoxide. Addition of DMSO in the reaction at a fi...
Keywords: AGE, Amplification, Buffers, Diagnostic, Electrophoresis, Evaporation, Gas chromatography, Gel electrophoresis, Melting, Nucleic acid annealing, Nucleic acid denaturation, Phase transitions, Polymerase chain reaction, Size-exclusion chromatography, Solvents

Reaction Optimization Reliable amplification of long DNA sequences requires: 1) effective denaturation of DNA template, 2) adequate extension times to produce large products and 3) protection of targ...
Keywords: AGE, Amplification, Buffers, Diagnostic, Electrophoresis, Evaporation, Gas chromatography, Gel electrophoresis, Melting, Nucleic acid annealing, Nucleic acid denaturation, Phase transitions, Polymerase chain reaction, Size-exclusion chromatography, Solvents

Every precaution should be taken to avoid contamination of reagents with unknown/unwanted DNA. This includes the following:
Keywords: AGE, Amplification, Buffers, Electrophoresis, Evaporation, Gas chromatography, Gel electrophoresis, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction

In the PCR Technologies Guide, the requisite components and quality control requirements for qPCR experiments were described in detail. With those in mind, the following is a protocol that can be use...
Keywords: Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography

These steps are presented below in greater detail along with materials and reagent selection tips. This is a basic PCR protocol using Taq DNA polymerase.
Keywords: AGE, Amplification, Electrophoresis, Evaporation, Gel electrophoresis, PAGE, Polymerase chain reaction, Sequencing

MTP™ Taq DNA Polymerase is a recombinant thermostable enzyme from Thermus aquaticus expressed in E. coli and purified using a proprietary process to minimize levels of contaminating DNA. The enzyme h...
Keywords: AGE, Amplification, Electrophoresis, Evaporation, Gel electrophoresis, Gene expression, PAGE, Polymerase chain reaction

Optimization of qPCR conditions is important for the development of a robust assay. Indications of poor optimization are a lack of reproducibility between replicates as well as inefficient and insens...
Keywords: Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography

In recent years Quantitative PCR has reached a level of sensitivity, accuracy, and ease to support use as a routine assay for measuring gene level expression. The field of cancer research is currentl...
Keywords: Amplification, Cancer, Clinical, Diagnostic, Gas chromatography, Gene expression, Genetic, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Polymorphisms, Sample preparations, Size-exclusion chromatography

In some cases it is preferable to measure the target transcript directly, without preparing cDNA from the entire RNA sample. Such situations may include measurements on highly degraded RNA or when th...
Keywords: Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography, Transcription

In the example given below, the primer concentrations can be adjusted according to the results of optimization procedures (see Primer Concentration Optimization, Primer Optimization Using Temperature...
Keywords: Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography

The SPUD assay is one option for identification of inhibitors that may be present in RNA or DNA samples. The assay is particularly useful when a large number of samples are to be analyzed or when tar...
Keywords: Amplification, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography

The SYBR Green Extract-N-Amp Plant PCR Kit contains the reagents needed for rapid extraction, amplification and detection of genomic DNA from plant leaves. DNA is rapidly extracted from a piece of le...
Keywords: Amplification, Degradations, Diagnostic, Gas chromatography, Metabolites, Nucleic acid annealing, Nucleic acid denaturation, Nucleic acid hybridization, Phase transitions, Polymerase chain reaction, Precipitation, Purification, Size-exclusion chromatography

Although quantitative PCR uses the same basic concept as traditional PCR, the reactions differ in that the amplicons are generally smaller and are detected indirectly using an additional dye or label...
Keywords: Gene expression, Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography

Reverse transcription (RT) is the process of converting RNA to cDNA using a reverse transcriptase enzyme and dNTPs.
Keywords: Amplification, Polymerase chain reaction, Polymerase chain reaction - quantitative, Transcription

The 3’/5’ integrity assay is a potential first step in the identification of RNA degradation. The assay is particularly useful when a large number of samples are to be analyzed or when the degradatio...
Keywords: Amplification, Degradations, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography, Transcription

Technology Overview Assay Considerations Methods of Quantification Equipment & Supplies PCR Mix Selection Guide Protocol Troubleshooting Materials References
Keywords: AGE, Amplification, Buffers, Degradations, Electrophoresis, Enzyme activity, Gas chromatography, Gel electrophoresis, Gene expression, Genetic, Melting, Microarray Analysis, Nucleic acid annealing, Nucleic acid denaturation, Nucleic acid hybridization, Polymerase chain reaction, Polymerase chain reaction - quantitative, Polymorphisms, Purification, Sample preparations, Size-exclusion chromatography, Solvents, Titrations

Once an assay has been optimized, it is important to verify the reaction efficiency. This information is important when reporting and comparing assays. In this example protocol, the assay efficiency ...
Keywords: Amplification, Gene expression, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography, Transcription

Measuring a target quantity relative to one or more stable reference genes using SYBR Green I dye detection is a common application of qPCR. Below is a standard protocol that can be adapted to specif...
Keywords: Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography, Transcription

The most common application for qPCR is the measurement of a gene transcript or copy number quantity relative to one or more reference genes using probe detection. The reactions may be designed such ...
Keywords: Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography, Transcription

Analysis of gene expression data requires a stable reference or loading control. This reference is usually one or more reference genes. Suitable reference genes are those which are unaffected by diff...
Keywords: Gene expression, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography, Transcription

In the PCR Technologies Guide, the requisite components and quality control requirements for qPCR experiments were described in detail. With those in mind, the following is a protocol that can be use...
Keywords: Nucleic acid annealing, Nucleic acid denaturation, Polymerase chain reaction, Polymerase chain reaction - quantitative, Size-exclusion chromatography

We offer a wide variety of PCR enzymes, master mixes, and PCR protocols to meet your experimental needs for routine PCR, qPCR, or RT-PCR. Our PCR Selection Guide features various filters to sort by, ...
Keywords: Genomics, Polymerase chain reaction, Polymerase chain reaction - quantitative