Protocols
Amplification of Damaged DNA with Restorase DNA Polymerase (R1028)
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
Amplification of Genomic DNA using REDTaq DNA Polymerase
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
Antibody-Enzyme Mediated Hot Start PCR Protocol
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
End Point PCR Protocol for Long and Accurate DNA Amplification
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
Gradient PCR For Optimization | Temperature Gradient Protocol
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
Hot Start Taq Polymerase Protocol to Reduce Non-Specific Amplification
As PCR reactions sit at room temperature, during assay setup, nonspecific amplification can occur via:
Keywords: AGE, Amplification, Electrophoresis, Gel electrophoresis, Gene expression, Nucleic acid annealing, Polymerase chain reaction, Size-exclusion chromatography
Kicqstart SYBR qPCR | Universal SYBR Green qPCR Protocol
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
Long and Accurate PCR Amplification of DNA with RedAccuTaq D4812 Protocol
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
Multiplex qPCR Protocol
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
MystiCq® MicroRNA® Quantitation System
Technology Overview Workflow Background Protocols miRNA isolation cDNA synthesis miRNA qPCR Advanced information Product selection guides Troubleshooting
Keywords: AGE, Amplification, Buffers, Cell disruption, Centrifugation, Degradations, Diagnostic, Digestions, Diseases, Electrophoresis, Gel electrophoresis, Gene expression, Homogenization, Microarray Analysis, Molecular biology, Nucleic acid annealing, Nucleic acid denaturation, PAGE, Polymerase chain reaction, Polymerase chain reaction - quantitative, Precipitation, Purification, RNA purification, Sample preparations, Separation, Solvents, Titrations
PCR Protocol | Standard PCR Protocol
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
PCR amplification of Bacterial DNA with low contamination using MTP™ Taq DNA Polymerase
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
Primer Concentration Optimization Protocol
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
Reverse Transcription Protocol (One-step Probe Detection)
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
Reverse Transcription Protocol (One-step SYBR Green I Dye Detection)
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
SPUD Assay for Detection of Assay Inhibitors Protocol
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
SYBR® Green I Dye Quantitative PCR Protocol
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
Standard Reverse Transcription Protocol (Two-step)
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' Assay for Analysis of RNA Integrity Protocol
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
Universal SYBR Green qPCR Protocol
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
dNTP Mediated Hot Start PCR Protocol
Hot Start dNTPs are modified with a thermolabile protecting group at the 3’ terminus. The presence of this modification blocks nucleotide incorporation by DNA polymerase until the nucleotide protecti...
Keywords: AGE, Amplification, Buffers, Centrifugation, Degradations, Electrophoresis, Gel electrophoresis, Nucleic acid denaturation, Polymerase chain reaction, Purification, Size-exclusion chromatography, Transcription
qPCR Efficiency Determination Protocol
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
qPCR Gene Expression/Copy Number Analysis Using SYBR Green I Dye Detection Protocol
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
qPCR Gene Expression/Copy Number/SNP Analysis Using Probe Detection Protocol
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
qPCR Reference Gene Selection Protocol
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
qPCR Using a Single Detection Probe Protocol
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
