Real-time PCR allows for the visualization and quantification of PCR reactions in real-time. It works by using fluorescent dyes that bind to double-stranded DNA and emit light during the PCR process, allowing researchers to measure increasing amounts of amplified DNA over successive cycles. Real-time PCR instruments contain a thermal cycler, optical module to detect fluorescence, and software to analyze the fluorescent data and calculate quantification values. The data appears as fluorescence levels over cycle number, and can be analyzed to determine starting template quantities or detect different DNA sequences through melt curve analysis.
Real-Time PCR Basics the polymerase chain reaction is process of amplificati...Sagarsharma899785
This document provides an overview of real-time PCR, including its advantages over traditional endpoint PCR and how it allows for quantitative analysis. Real-time PCR detects PCR product amplification throughout the reaction in real-time using fluorescent dyes. It generates data on DNA copy numbers during each cycle that can be analyzed to determine starting template quantities based on cycle thresholds. Real-time PCR is used for applications like gene expression analysis, disease diagnosis, food testing, and more.
The document discusses polymerase chain reaction (PCR) and its use for detecting COVID-19 through RT-PCR testing. RT-PCR is considered the gold standard for detecting viruses like SARS-CoV-2 due to its rapid detection, high sensitivity, and specificity. The document outlines the RT-PCR testing process, which involves collecting a sample, extracting RNA from the sample, converting the RNA to cDNA, and amplifying the cDNA using fluorescent dyes to detect the presence of the virus. Real-time PCR allows visualization and quantification of PCR product accumulation in real-time by using fluorescent dyes that bind to DNA.
This document discusses real-time PCR, including its advantages over traditional PCR, the theory behind how it works, different chemistries that can be used like SYBR Green and TaqMan, and methods for quantification like standard curves and the delta delta Ct method. Real-time PCR allows amplification to be monitored in real-time, has a wider dynamic range and does not require post-PCR processing. It uses fluorescent dyes and probes and software to analyze template concentration from fluorescence detection during amplification cycles. Common housekeeping genes used for normalization include GAPDH, beta-actin, and ribosomal proteins.
Real-time PCR (polymerase chain reaction) allows for amplification and quantification of DNA during the PCR process through the use of fluorescent probes such as TaqMan probes or SYBR Green. It provides advantages over conventional PCR such as faster results, higher sensitivity in detecting small changes in DNA amounts, and the ability to quantify initial template concentrations through analysis of threshold cycle values. Common applications include detection of gene expression, viral load quantification, and molecular diagnostics.
Real-time PCR allows for the continuous collection of fluorescent data during the PCR process, allowing for quantification of the amount of PCR product accumulated in each cycle. It provides advantages over conventional PCR such as increased precision, sensitivity, and automation. Various chemistries can be used including SYBR Green, TaqMan probes, molecular beacons, and scorpion primers, which rely on fluorescent dyes and quenchers. Real-time PCR finds applications in gene expression analysis, pathogen detection, and DNA damage measurement by allowing quantitative analysis.
Real-time PCR allows for the quantification of PCR products in real time during the amplification process. It involves the use of fluorescent dyes or probes that increase in fluorescence as more PCR products are generated. Common fluorescent dyes used include SYBR Green and various hydrolysis probes like TaqMan probes. Real-time PCR provides advantages over conventional PCR such as higher sensitivity, quantification capabilities, and automation. It has various applications including disease diagnosis, gene expression analysis, and SNP genotyping.
Real-time PCR allows the amplification of specific DNA fragments to be visualized in real time as the reaction progresses. It allows minute amounts of DNA sequences to be measured in a sample. Quantitative real-time PCR converts the fluorescent signals from each reaction into a numerical value for each sample. TaqMan probes are widely used in real-time PCR assays to monitor reactions and quantify specific DNA sequences in applications like gene expression analysis, viral load detection, and SNP genotyping.
Real-time PCR is a technique that monitors DNA amplification during the PCR process in real-time using fluorescence detection. It allows for both quantification of DNA present and detection of DNA amplification as it occurs. Real-time PCR has advantages over traditional PCR such as higher sensitivity, specificity, and ability to provide quantitative results. It uses sequence-specific DNA probes labeled with fluorescent dyes and quenchers to detect amplification of target DNA sequences. Data analysis can provide both absolute and relative quantification of DNA targets. Real-time PCR has many applications including gene expression analysis, disease diagnosis, and food and environmental testing.
Real-Time PCR Basics the polymerase chain reaction is process of amplificati...Sagarsharma899785
This document provides an overview of real-time PCR, including its advantages over traditional endpoint PCR and how it allows for quantitative analysis. Real-time PCR detects PCR product amplification throughout the reaction in real-time using fluorescent dyes. It generates data on DNA copy numbers during each cycle that can be analyzed to determine starting template quantities based on cycle thresholds. Real-time PCR is used for applications like gene expression analysis, disease diagnosis, food testing, and more.
The document discusses polymerase chain reaction (PCR) and its use for detecting COVID-19 through RT-PCR testing. RT-PCR is considered the gold standard for detecting viruses like SARS-CoV-2 due to its rapid detection, high sensitivity, and specificity. The document outlines the RT-PCR testing process, which involves collecting a sample, extracting RNA from the sample, converting the RNA to cDNA, and amplifying the cDNA using fluorescent dyes to detect the presence of the virus. Real-time PCR allows visualization and quantification of PCR product accumulation in real-time by using fluorescent dyes that bind to DNA.
This document discusses real-time PCR, including its advantages over traditional PCR, the theory behind how it works, different chemistries that can be used like SYBR Green and TaqMan, and methods for quantification like standard curves and the delta delta Ct method. Real-time PCR allows amplification to be monitored in real-time, has a wider dynamic range and does not require post-PCR processing. It uses fluorescent dyes and probes and software to analyze template concentration from fluorescence detection during amplification cycles. Common housekeeping genes used for normalization include GAPDH, beta-actin, and ribosomal proteins.
Real-time PCR (polymerase chain reaction) allows for amplification and quantification of DNA during the PCR process through the use of fluorescent probes such as TaqMan probes or SYBR Green. It provides advantages over conventional PCR such as faster results, higher sensitivity in detecting small changes in DNA amounts, and the ability to quantify initial template concentrations through analysis of threshold cycle values. Common applications include detection of gene expression, viral load quantification, and molecular diagnostics.
Real-time PCR allows for the continuous collection of fluorescent data during the PCR process, allowing for quantification of the amount of PCR product accumulated in each cycle. It provides advantages over conventional PCR such as increased precision, sensitivity, and automation. Various chemistries can be used including SYBR Green, TaqMan probes, molecular beacons, and scorpion primers, which rely on fluorescent dyes and quenchers. Real-time PCR finds applications in gene expression analysis, pathogen detection, and DNA damage measurement by allowing quantitative analysis.
Real-time PCR allows for the quantification of PCR products in real time during the amplification process. It involves the use of fluorescent dyes or probes that increase in fluorescence as more PCR products are generated. Common fluorescent dyes used include SYBR Green and various hydrolysis probes like TaqMan probes. Real-time PCR provides advantages over conventional PCR such as higher sensitivity, quantification capabilities, and automation. It has various applications including disease diagnosis, gene expression analysis, and SNP genotyping.
Real-time PCR allows the amplification of specific DNA fragments to be visualized in real time as the reaction progresses. It allows minute amounts of DNA sequences to be measured in a sample. Quantitative real-time PCR converts the fluorescent signals from each reaction into a numerical value for each sample. TaqMan probes are widely used in real-time PCR assays to monitor reactions and quantify specific DNA sequences in applications like gene expression analysis, viral load detection, and SNP genotyping.
Real-time PCR is a technique that monitors DNA amplification during the PCR process in real-time using fluorescence detection. It allows for both quantification of DNA present and detection of DNA amplification as it occurs. Real-time PCR has advantages over traditional PCR such as higher sensitivity, specificity, and ability to provide quantitative results. It uses sequence-specific DNA probes labeled with fluorescent dyes and quenchers to detect amplification of target DNA sequences. Data analysis can provide both absolute and relative quantification of DNA targets. Real-time PCR has many applications including gene expression analysis, disease diagnosis, and food and environmental testing.
This document discusses real-time reverse transcription polymerase chain reaction (rt-PCR). It explains that rt-PCR monitors the amplification of a targeted nucleic acid molecule during PCR reactions in real time, unlike conventional PCR which detects products at the end. The document outlines the basic process of rt-PCR including setting up target genes with different fluorescent labels, performing the rt-PCR with a thermal cycler, and interpreting results based on cycle threshold values.
The document discusses reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR). RT-PCR is used to amplify RNA templates by first reverse transcribing the RNA to cDNA. There are different strategies for first-strand cDNA synthesis such as using random primers or oligo dT primers. Real-time qPCR allows for monitoring of the PCR reaction in real-time using fluorescent dyes or probes. Common probes include SYBR green, TaqMan probes, and molecular beacons. The threshold cycle (CT) value provides a quantitative measure related to the starting quantity of nucleic acid. Real-time qPCR has various applications including clinical diagnosis, DNA sequencing, comparative genomic studies,
Real Time PCR, also known as quantitative PCR (qPCR), allows for the amplification and quantification of specific DNA sequences in real time as the reaction progresses after each cycle. It involves monitoring fluorescence levels after each cycle to determine the amount of PCR product accumulated. There are two main chemistries used - SYBR Green, which binds nonspecifically to double stranded DNA, and TaqMan probes, which provide sequence-specific detection. Real Time PCR has various applications including gene expression analysis, pathogen detection, and quantification of DNA or RNA targets.
This document provides an outline and overview of real-time PCR. It begins with basics and definitions of real-time PCR, listing its advantages over conventional PCR. Principles of real-time PCR are explained, along with useful terms. The document then discusses real-time PCR chemistry, explaining fluorescence dyes and probes used including SYBR Green, TaqMan probes, and molecular beacons. Instruments, assay design, data analysis, and troubleshooting are also outlined.
This document provides an overview of real-time PCR. It discusses the basics of real-time PCR including how it works, terminology, and uses. It also covers experimental design considerations like singleplex vs multiplex assays and fluorescent chemistry selection. Practical aspects are reviewed like plate loading, standard curves, and melting curves. Data analysis techniques like absolute and relative quantification and gene expression analysis are also summarized.
The document provides information on PCR and RT-PCR including definitions, components, steps, types, and applications. PCR is described as a technique for amplifying a single DNA template using thermal cycling. It requires a DNA template, primers, Taq polymerase, dNTPs, and buffer. The main steps are denaturation of the DNA, annealing of primers, and elongation. RT-PCR is described as a technique for amplifying RNA using reverse transcriptase to generate cDNA, which is then amplified using PCR. Applications described include disease diagnosis, forensics, paternity testing, and detecting infections.
This document reviews the basic principles of real-time quantitative PCR. It discusses how real-time PCR allows sensitive and reproducible quantification of nucleic acids during PCR amplification by detecting fluorescent signals in real time. The document describes various chemistries used in real-time PCR including SYBR Green, hydrolysis probes, molecular beacons, and explains the quantification method. Real-time PCR provides accurate quantification during the exponential phase of amplification by measuring threshold cycle (Ct) values, before the reaction reaches plateau. The technique has many applications in molecular diagnostics and gene expression analysis.
PCR can be used for cloning, DNA fingerprinting, gene expression analysis, DNA sequencing, and more. It works by amplifying a specific region of DNA through repeated heating and cooling cycles. This allows very small amounts of DNA to be exponentially multiplied, enabling various applications. PCR was invented in 1983 by Kary Mullis and revolutionized molecular biology.
BME 302 Cellular Engineering Common Cell & Molecu.docxhartrobert670
BME 302
Cellular Engineering
Common Cell &
Molecular Biology
Assays
1
Common Cell & Molecular
Biology Assays
u Why ?
time=0
signal
Time=t1
?
Qualitatively vs. Quantitatively
What is the
specific
effect of the
trigger e.g.
CELL
PHENOTYPE?
How did this
happen e.g.
Mechanism –
specific cell
transduction
pathway?
Chemical
(GFs)
Mechanical
(pressure)
Common Cell & Molecular
Biology Assays
u Why ?
3
Signals:
Gene expression
CELL
PHENOTYPE
Mechanism:
Specific
signaling
pathway
Common Cell & Molecular
Biology Assays
u How do you characterize the CELL PHENOTYPE?
u Quantification of gene expression through mRNA
quantification
u PCR (end-point and real-time)
u Quantification of proteins (cell epitopes/markers,
secreted cytokines)
u In situ (tissue sections / cells) – Immunostaining
u Microscopy for 2D vs. 3D imaging
u In solutions (supernatants / tissue lysates) –
ELISA
u On the cell surface – FACS
4
Different stages
Ex. Bronchial Epithelial Cell
Differentiation
5
Immunostaining
ELISA
Histochemistry
Basal cells
Epithelial cells
Ex. Long-term self-renewal of
human pluripotent stem cells on
human recombinant laminin511
6
Nature Biotechnology
Volume:
28,
Pages:
611–615
Year published:
(2010)
Nature Biotechnology 28, 611-615 (2010)
FACS
Immunostaining End-point PCR
Real-time PCR
Western Blot
To replace MG
M
G
LN
Gene Transcription & Protein Translation
DNA –> RNA -> Protein
7
Secreted Protein
Internal Protein
PCR
Polymerase Chain Reaction
u Why PCR ?
u Produces many DNA sequence copies without the need of
host cloning (genetic engineering lecture)
u Amplifies known DNA sequences from mRNA reverse
transcription for analysis of gene expression
u Extract mRNA -> rt in cDNA
8
PCR
Polymerase Chain Reaction
9
Cycle 3 Cycle 1
Produces 2 molecules Produces 4
molecules
Produces 8
molecules
Cycle 2
DNA containing
target sequence
to be amplified
Target
sequence
Target
sequence
Target
sequence Template
DNA
primer
DNA
primer
Template
New
DNA
New
DNA
These 2
molecules
match
target DNA
sequence
DNA
primers
DNA
primers
PCR
Polymerase Chain Reaction
u What do you need ?
10
https://www.youtube.com/watch?
v=2KoLnIwoZKU
Traditional end-point PCR
11
Agarose
gel
Buffer
solution
Gel box
Well in gel for
placing DNA
sample
PCR
products
already
loaded to
wells
+
–
+
–
Figure 18.7: Research Method.
Separation of DNA Fragments by Agarose Gel Electrophoresis
Micropipettor
adding marker
DNA fragments
to well
Traditional end-point PCR
Figure 18.7: Research Method.
Separation of DNA Fragments by Agarose Gel Electrophoresis
Lane with
marker DNA
fragments
Quantification of end-point
PCR
13
Limitations of End-Point PCR
u Poor Precision
u Low sens ...
1073958 wp guide-develop-pcr_primers_1012Elsa von Licy
methods analyze the exponential phase of individual amplification
1. The document outlines guidelines for developing high-quality real-time PCR primers based on lessons from designing assays for over 14,000 genes.
plots. Regardless of the method, efficiencies between 90-110
2. Key factors in primer design include thermodynamic properties, specificity testing to ensure a single amplicon, and verification of high amplification efficiency and reproducibility.
percent are generally acceptable for accurate analysis by the
3. Wet-bench testing of primers is crucial to validate specificity with single peak melt curves and correct sized products on gels, as well as high efficiency.
∆∆CT method.
The document discusses polymerase chain reaction (PCR), including its history, principles, types, applications, and future. It was invented in 1984 as a way to amplify DNA fragments in the laboratory. PCR works by heating and cooling DNA to make millions of copies of a target sequence. It has many applications in medicine, infectious disease detection, forensics, and research. Quantitative PCR allows measuring DNA quantities and is commonly used to detect gene expression levels. The future of PCR includes more sensitive techniques like immunoliposome-PCR.
This document provides an overview of real-time PCR, including commonly used formats, the basic steps involved in real-time PCR, and an overview of key reaction components. Real-time PCR allows for quantification of DNA or RNA sequences by measuring fluorescent signals during each PCR cycle. It discusses single-tube assays, multi-well plates, and array cards as common formats. The basic steps of real-time PCR involve denaturation, annealing of primers, and extension by DNA polymerase in each cycle. Key components that can affect results are the DNA polymerase, reverse transcriptase, dNTPs, magnesium concentration, and template quality and quantity.
Real-time PCR is a technique that can detect and quantify DNA amplification as the PCR reaction occurs in real-time. It allows quantification of very small amounts of DNA (<1 ng). The document discusses the basic principles and steps of real-time PCR including RNA isolation, cDNA synthesis, quantitative PCR, data analysis and applications such as gene expression analysis, viral load measurement, and pathogen detection. Real-time PCR provides accurate, specific and sensitive quantification of nucleic acids.
The document describes the polymerase chain reaction (PCR) technique for amplifying DNA. It discusses the basic components and steps of PCR, including denaturation, annealing and extension. It also describes different PCR types such as nested PCR, RT-PCR, and applications in clinical diagnosis, forensics and research. PCR is a powerful technique for amplifying specific DNA regions, enabling various downstream applications.
Real Time PCR allows for detection and quantification of DNA as amplification occurs. It monitors fluorescence at each cycle to measure DNA accumulation. There are two main types of instrumentation - two-step qRT-PCR which involves reverse transcription followed by PCR, and one-step which combines these steps. Detection relies on fluorescent dyes like SYBR Green or target-specific Taqman probes. Real Time PCR provides advantages over conventional PCR like not requiring gels and being faster and less complex for quantification.
REAL-TIME PCR.pptx by UMNA FATIMA- BIOMEDumnajmi123
his PowerPoint presentation serves as a comprehensive guide to understanding Reverse Transcription Polymerase Chain Reaction (RT-PCR), a fundamental technique in molecular biology research and diagnostics. The presentation begins with an introduction to the principle of RT-PCR, elucidating its significance in analyzing RNA molecules and amplifying specific sequences of interest. It then delves into the key components and steps involved in RT-PCR, including RNA extraction, cDNA synthesis, PCR amplification, and real-time detection. Through informative slides, the audience will gain insights into the applications of RT-PCR in various fields such as disease diagnosis, gene expression analysis, and environmental monitoring. Additionally, the presentation addresses quality control measures, data analysis techniques, and offers case studies illustrating real-world applications of RT-PCR. Furthermore, it discusses future perspectives and emerging trends in RT-PCR technology, providing a forward-looking view of its potential advancements and implications. With a focus on clarity and comprehensiveness, this presentation aims to equip the audience with a thorough understanding of RT-PCR and its diverse applications in molecular biology.
This document describes how real-time PCR can be used to validate microarray data. Real-time PCR provides a quantitative and sensitive method for confirming changes in gene expression observed in microarray experiments. The document outlines a protocol for designing and running a real-time PCR experiment to validate a specific result from a microarray experiment showing increased expression of the TNFAIP3 gene in response to TNFα treatment. Key steps in the protocol include performing reverse transcription of RNA to generate cDNA, setting up a standard curve and controls, and analyzing the real-time PCR data to calculate fold-changes in gene expression.
real-time PCR .... by aqee-lhadithe - sem ivAqeelhadithe
Real-time PCR (qPCR) allows for detection of PCR products during the reaction, not just at the end. It uses a fluorescent dye like SYBR Green or hydrolysis probes like TaqMan probes.
The threshold cycle (Ct) value indicates the cycle when fluorescence crosses a threshold. Lower Ct values mean a higher initial amount of target DNA.
Primer and probe design is important for specificity and efficiency. Primers should be around 18-25 nucleotides, have 50-60% GC content, avoid repeats and polymorphisms, and target intron-exon boundaries when possible.
PCR is a technique used to amplify specific DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to separate the DNA strands and allow primers to bind. The primers and DNA polymerase then work to replicate the target DNA region, doubling the amount with each cycle. PCR is useful for cloning genes, detecting genetic diseases and mutations, identifying microorganisms, and analyzing gene expression patterns. It has many applications in research, forensics, and medical diagnosis.
This document discusses real-time reverse transcription polymerase chain reaction (rt-PCR). It explains that rt-PCR monitors the amplification of a targeted nucleic acid molecule during PCR reactions in real time, unlike conventional PCR which detects products at the end. The document outlines the basic process of rt-PCR including setting up target genes with different fluorescent labels, performing the rt-PCR with a thermal cycler, and interpreting results based on cycle threshold values.
The document discusses reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR (qPCR). RT-PCR is used to amplify RNA templates by first reverse transcribing the RNA to cDNA. There are different strategies for first-strand cDNA synthesis such as using random primers or oligo dT primers. Real-time qPCR allows for monitoring of the PCR reaction in real-time using fluorescent dyes or probes. Common probes include SYBR green, TaqMan probes, and molecular beacons. The threshold cycle (CT) value provides a quantitative measure related to the starting quantity of nucleic acid. Real-time qPCR has various applications including clinical diagnosis, DNA sequencing, comparative genomic studies,
Real Time PCR, also known as quantitative PCR (qPCR), allows for the amplification and quantification of specific DNA sequences in real time as the reaction progresses after each cycle. It involves monitoring fluorescence levels after each cycle to determine the amount of PCR product accumulated. There are two main chemistries used - SYBR Green, which binds nonspecifically to double stranded DNA, and TaqMan probes, which provide sequence-specific detection. Real Time PCR has various applications including gene expression analysis, pathogen detection, and quantification of DNA or RNA targets.
This document provides an outline and overview of real-time PCR. It begins with basics and definitions of real-time PCR, listing its advantages over conventional PCR. Principles of real-time PCR are explained, along with useful terms. The document then discusses real-time PCR chemistry, explaining fluorescence dyes and probes used including SYBR Green, TaqMan probes, and molecular beacons. Instruments, assay design, data analysis, and troubleshooting are also outlined.
This document provides an overview of real-time PCR. It discusses the basics of real-time PCR including how it works, terminology, and uses. It also covers experimental design considerations like singleplex vs multiplex assays and fluorescent chemistry selection. Practical aspects are reviewed like plate loading, standard curves, and melting curves. Data analysis techniques like absolute and relative quantification and gene expression analysis are also summarized.
The document provides information on PCR and RT-PCR including definitions, components, steps, types, and applications. PCR is described as a technique for amplifying a single DNA template using thermal cycling. It requires a DNA template, primers, Taq polymerase, dNTPs, and buffer. The main steps are denaturation of the DNA, annealing of primers, and elongation. RT-PCR is described as a technique for amplifying RNA using reverse transcriptase to generate cDNA, which is then amplified using PCR. Applications described include disease diagnosis, forensics, paternity testing, and detecting infections.
This document reviews the basic principles of real-time quantitative PCR. It discusses how real-time PCR allows sensitive and reproducible quantification of nucleic acids during PCR amplification by detecting fluorescent signals in real time. The document describes various chemistries used in real-time PCR including SYBR Green, hydrolysis probes, molecular beacons, and explains the quantification method. Real-time PCR provides accurate quantification during the exponential phase of amplification by measuring threshold cycle (Ct) values, before the reaction reaches plateau. The technique has many applications in molecular diagnostics and gene expression analysis.
PCR can be used for cloning, DNA fingerprinting, gene expression analysis, DNA sequencing, and more. It works by amplifying a specific region of DNA through repeated heating and cooling cycles. This allows very small amounts of DNA to be exponentially multiplied, enabling various applications. PCR was invented in 1983 by Kary Mullis and revolutionized molecular biology.
BME 302 Cellular Engineering Common Cell & Molecu.docxhartrobert670
BME 302
Cellular Engineering
Common Cell &
Molecular Biology
Assays
1
Common Cell & Molecular
Biology Assays
u Why ?
time=0
signal
Time=t1
?
Qualitatively vs. Quantitatively
What is the
specific
effect of the
trigger e.g.
CELL
PHENOTYPE?
How did this
happen e.g.
Mechanism –
specific cell
transduction
pathway?
Chemical
(GFs)
Mechanical
(pressure)
Common Cell & Molecular
Biology Assays
u Why ?
3
Signals:
Gene expression
CELL
PHENOTYPE
Mechanism:
Specific
signaling
pathway
Common Cell & Molecular
Biology Assays
u How do you characterize the CELL PHENOTYPE?
u Quantification of gene expression through mRNA
quantification
u PCR (end-point and real-time)
u Quantification of proteins (cell epitopes/markers,
secreted cytokines)
u In situ (tissue sections / cells) – Immunostaining
u Microscopy for 2D vs. 3D imaging
u In solutions (supernatants / tissue lysates) –
ELISA
u On the cell surface – FACS
4
Different stages
Ex. Bronchial Epithelial Cell
Differentiation
5
Immunostaining
ELISA
Histochemistry
Basal cells
Epithelial cells
Ex. Long-term self-renewal of
human pluripotent stem cells on
human recombinant laminin511
6
Nature Biotechnology
Volume:
28,
Pages:
611–615
Year published:
(2010)
Nature Biotechnology 28, 611-615 (2010)
FACS
Immunostaining End-point PCR
Real-time PCR
Western Blot
To replace MG
M
G
LN
Gene Transcription & Protein Translation
DNA –> RNA -> Protein
7
Secreted Protein
Internal Protein
PCR
Polymerase Chain Reaction
u Why PCR ?
u Produces many DNA sequence copies without the need of
host cloning (genetic engineering lecture)
u Amplifies known DNA sequences from mRNA reverse
transcription for analysis of gene expression
u Extract mRNA -> rt in cDNA
8
PCR
Polymerase Chain Reaction
9
Cycle 3 Cycle 1
Produces 2 molecules Produces 4
molecules
Produces 8
molecules
Cycle 2
DNA containing
target sequence
to be amplified
Target
sequence
Target
sequence
Target
sequence Template
DNA
primer
DNA
primer
Template
New
DNA
New
DNA
These 2
molecules
match
target DNA
sequence
DNA
primers
DNA
primers
PCR
Polymerase Chain Reaction
u What do you need ?
10
https://www.youtube.com/watch?
v=2KoLnIwoZKU
Traditional end-point PCR
11
Agarose
gel
Buffer
solution
Gel box
Well in gel for
placing DNA
sample
PCR
products
already
loaded to
wells
+
–
+
–
Figure 18.7: Research Method.
Separation of DNA Fragments by Agarose Gel Electrophoresis
Micropipettor
adding marker
DNA fragments
to well
Traditional end-point PCR
Figure 18.7: Research Method.
Separation of DNA Fragments by Agarose Gel Electrophoresis
Lane with
marker DNA
fragments
Quantification of end-point
PCR
13
Limitations of End-Point PCR
u Poor Precision
u Low sens ...
1073958 wp guide-develop-pcr_primers_1012Elsa von Licy
methods analyze the exponential phase of individual amplification
1. The document outlines guidelines for developing high-quality real-time PCR primers based on lessons from designing assays for over 14,000 genes.
plots. Regardless of the method, efficiencies between 90-110
2. Key factors in primer design include thermodynamic properties, specificity testing to ensure a single amplicon, and verification of high amplification efficiency and reproducibility.
percent are generally acceptable for accurate analysis by the
3. Wet-bench testing of primers is crucial to validate specificity with single peak melt curves and correct sized products on gels, as well as high efficiency.
∆∆CT method.
The document discusses polymerase chain reaction (PCR), including its history, principles, types, applications, and future. It was invented in 1984 as a way to amplify DNA fragments in the laboratory. PCR works by heating and cooling DNA to make millions of copies of a target sequence. It has many applications in medicine, infectious disease detection, forensics, and research. Quantitative PCR allows measuring DNA quantities and is commonly used to detect gene expression levels. The future of PCR includes more sensitive techniques like immunoliposome-PCR.
This document provides an overview of real-time PCR, including commonly used formats, the basic steps involved in real-time PCR, and an overview of key reaction components. Real-time PCR allows for quantification of DNA or RNA sequences by measuring fluorescent signals during each PCR cycle. It discusses single-tube assays, multi-well plates, and array cards as common formats. The basic steps of real-time PCR involve denaturation, annealing of primers, and extension by DNA polymerase in each cycle. Key components that can affect results are the DNA polymerase, reverse transcriptase, dNTPs, magnesium concentration, and template quality and quantity.
Real-time PCR is a technique that can detect and quantify DNA amplification as the PCR reaction occurs in real-time. It allows quantification of very small amounts of DNA (<1 ng). The document discusses the basic principles and steps of real-time PCR including RNA isolation, cDNA synthesis, quantitative PCR, data analysis and applications such as gene expression analysis, viral load measurement, and pathogen detection. Real-time PCR provides accurate, specific and sensitive quantification of nucleic acids.
The document describes the polymerase chain reaction (PCR) technique for amplifying DNA. It discusses the basic components and steps of PCR, including denaturation, annealing and extension. It also describes different PCR types such as nested PCR, RT-PCR, and applications in clinical diagnosis, forensics and research. PCR is a powerful technique for amplifying specific DNA regions, enabling various downstream applications.
Real Time PCR allows for detection and quantification of DNA as amplification occurs. It monitors fluorescence at each cycle to measure DNA accumulation. There are two main types of instrumentation - two-step qRT-PCR which involves reverse transcription followed by PCR, and one-step which combines these steps. Detection relies on fluorescent dyes like SYBR Green or target-specific Taqman probes. Real Time PCR provides advantages over conventional PCR like not requiring gels and being faster and less complex for quantification.
REAL-TIME PCR.pptx by UMNA FATIMA- BIOMEDumnajmi123
his PowerPoint presentation serves as a comprehensive guide to understanding Reverse Transcription Polymerase Chain Reaction (RT-PCR), a fundamental technique in molecular biology research and diagnostics. The presentation begins with an introduction to the principle of RT-PCR, elucidating its significance in analyzing RNA molecules and amplifying specific sequences of interest. It then delves into the key components and steps involved in RT-PCR, including RNA extraction, cDNA synthesis, PCR amplification, and real-time detection. Through informative slides, the audience will gain insights into the applications of RT-PCR in various fields such as disease diagnosis, gene expression analysis, and environmental monitoring. Additionally, the presentation addresses quality control measures, data analysis techniques, and offers case studies illustrating real-world applications of RT-PCR. Furthermore, it discusses future perspectives and emerging trends in RT-PCR technology, providing a forward-looking view of its potential advancements and implications. With a focus on clarity and comprehensiveness, this presentation aims to equip the audience with a thorough understanding of RT-PCR and its diverse applications in molecular biology.
This document describes how real-time PCR can be used to validate microarray data. Real-time PCR provides a quantitative and sensitive method for confirming changes in gene expression observed in microarray experiments. The document outlines a protocol for designing and running a real-time PCR experiment to validate a specific result from a microarray experiment showing increased expression of the TNFAIP3 gene in response to TNFα treatment. Key steps in the protocol include performing reverse transcription of RNA to generate cDNA, setting up a standard curve and controls, and analyzing the real-time PCR data to calculate fold-changes in gene expression.
real-time PCR .... by aqee-lhadithe - sem ivAqeelhadithe
Real-time PCR (qPCR) allows for detection of PCR products during the reaction, not just at the end. It uses a fluorescent dye like SYBR Green or hydrolysis probes like TaqMan probes.
The threshold cycle (Ct) value indicates the cycle when fluorescence crosses a threshold. Lower Ct values mean a higher initial amount of target DNA.
Primer and probe design is important for specificity and efficiency. Primers should be around 18-25 nucleotides, have 50-60% GC content, avoid repeats and polymorphisms, and target intron-exon boundaries when possible.
PCR is a technique used to amplify specific DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to separate the DNA strands and allow primers to bind. The primers and DNA polymerase then work to replicate the target DNA region, doubling the amount with each cycle. PCR is useful for cloning genes, detecting genetic diseases and mutations, identifying microorganisms, and analyzing gene expression patterns. It has many applications in research, forensics, and medical diagnosis.
Histololgy of Female Reproductive System.pptxAyeshaZaid1
Dive into an in-depth exploration of the histological structure of female reproductive system with this comprehensive lecture. Presented by Dr. Ayesha Irfan, Assistant Professor of Anatomy, this presentation covers the Gross anatomy and functional histology of the female reproductive organs. Ideal for students, educators, and anyone interested in medical science, this lecture provides clear explanations, detailed diagrams, and valuable insights into female reproductive system. Enhance your knowledge and understanding of this essential aspect of human biology.
Promoting Wellbeing - Applied Social Psychology - Psychology SuperNotesPsychoTech Services
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These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
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2. Real-Time PCR
David A. Palmer, Ph.D.
Technical Support, Bio-Rad Laboratories
Adjunct Professor, Contra Costa College
3. Objectives This presentation will cover the
following topics:
• What is real-time PCR used for?
• How does real-time PCR work?
• What instruments are used?
• What does real-time data look like?
• How can the Crime Scene Invesigator
kit be used in a real-time setting?
5. What is
Real-Time
PCR?
The Polymerase Chain Reaction (PCR)
is a process for the amplification of
specific fragments of DNA.
Real-Time PCR a specialized technique
that allows a PCR reaction to be
visualized “in real time” as the
reaction progresses.
As we will see, Real-Time PCR allows
us to measure minute amounts of
DNA sequences in a sample!
6. What is
Real-Time
PCR used
for?
Real-Time PCR has become a cornerstone of
molecular biology:
• Gene expression analysis
–Cancer research
–Drug research
• Disease diagnosis and management
–Viral quantification
• Food testing
–Percent GMO food
• Animal and plant breeding
–Gene copy number
7. Real-Time
PCR in
Gene
Expression
Analysis
Example: BRCA1 Expression Profiling
BRCA1 is a gene involved in tumor suppression.
BRCA1 controls the expression of other genes.
In order to monitor level of expression of BRCA1,
real-time PCR is used.
DNA
mRNA
Protein
BRCA1
8. Real-Time
PCR in
Disease
Management
Example: HIV Treatment
Drug treatment for HIV infection often depends on
monitoring the “viral load”.
Real-Time PCR allows for direct measurement of the
amount of the virus RNA in the patient.
Virus
RNA
9. Real-Time
PCR in Food
Testing
Example: Determining percentage of GMO food
content
Determination of percent GMO food content
important for import / export regulations.
Labs use Real-Time PCR to measure amount of
transgenic versus wild-type DNA.
Seed
wt DNA
GMO DNA
15. How does
Real-Time
PCR work?
…So that’s how traditional PCR is usually presented.
In order to understand real-time PCR, let’s use a
“thought experiment”, and save all of the
calculations and formulas until later…
Most importantly, we’ll start by imagining the PCR
itself, and only then will we draw graphs to
illustrate what’s going on.
NO GRAPHS
(yet)
17. Imagining
Real-Time
PCR
What’s in our tube, at cycle number 25?
A soup of nucleotides, primers, template,
amplicons, enzyme, etc.
1,000,000 copies of the amplicon right now.
18. Imagining
Real-Time
PCR
How did we
get here?
What was it like last cycle, 24?
Almost exactly the same, except there were
only 500,000 copies of the amplicon.
And the cycle before that, 23?
Almost the same, but only 250,000 copies of
the amplicon.
And what about cycle 22?
Not a whole lot different. 125,000 copies of
the amplicon.
19. Imagining
Real-Time
PCR
How did we
get here?
If we were to graph the amount of DNA in our
tube, from the start until right now, at cycle
25, the graph would look like this:
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
0 5 10 15 20 25 30 35 40
20. Imagining
Real-Time
PCR
How did we
get here?
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
0 5 10 15 20 25 30 35 40
So, right now we’re at cycle 25 in a soup with
1,000,000 copies of the target.
What’s it going to be like after the next cycle,
in cycle 26?
?
21. Imagining
Real-Time
PCR
So where
are we
going?
What’s it going to be like after the next cycle, in cycle 26?
Probably there will be 2,000,000 amplicons.
And cycle 27?
Maybe 4,000,000 amplicons.
And at cycle 200?
In theory, there would be
1,000,000,000,000,000,000,000,000,000,000,000,000,000,000,0
00,000,000,000,000 amplicons…
Or 10^35 tonnes of DNA…
To put this in perspective, that would be equivalent to the weight of
ten billion planets the size of Earth!!!!
22. Imagining
Real-Time
PCR
So where
are we
going?
A clump of DNA the size of ten billion planets
won’t quite fit in our PCR tube anymore.
Realistically, at the chain reaction progresses,
it gets exponentially harder to find primers,
and nucleotides. And the polymerase is
wearing out.
So exponential growth does not go on
forever!
23. Imagining
Real-Time
PCR
So where
are we
going?
If we plot the amount of DNA in our tube
going forward from cycle 25, we see that it
actually looks like this:
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
25. Imagining
Real-Time
PCR
Measuring
Quantities
Let’s imagine that you start with four times as
much DNA as I do…picture our two tubes at
cycle 25 and work backwards a few cycles.
Cycle Me You
23 250,000 1,000,000
24 500,000 2,000,000
25 1,000,000 4,000,000
Cycle 25
26. Imagining
Real-Time
PCR
Measuring
Quantities
So, if YOU started with FOUR times as much
DNA template as I did…
…Then you’d reach 1,000,000 copies exactly
TWO cycles earlier than I would!
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
28. Imagining
Real-Time
PCR
Measuring
Quantities
What if YOU started with EIGHT times LESS DNA template
than I did?
You’d only have 125,000 copies right now at cycle 25…
And you’d reach 1,000,000 copies exactly THREE cycles
later than I would!
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
29. Imagining
Real-Time
PCR
Measuring
Quantities
We describe the position of the lines with a value that
represents the cycle number where the trace crosses an
arbitrary threshold.
This is called the “Ct Value”.
Ct values are directly related to the starting quantity of
DNA, by way of the formula:
Quantity = 2^Ct
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
0 5 10 15 20 25 30 35 40
23 25 28
Ct Values:
31. Imagining
Real-Time
PCR
Measuring
Quantities
There’s a DIRECT relationship between the
starting amount of DNA, and the cycle
number that you’ll reach an arbitrary number
of DNA copies (Ct value).
DNA amount ≈ 2 Cycle Number
C o p y N u m b e r v s. C t - Sta n d a r d C u r v e
y = -3 . 3 1 9 2 x + 3 9 . 7 7 2
R
2
= 0 .9 9 6 7
0
5
1 0
1 5
2 0
2 5
3 0
3 5
4 0
0 1 2 3 4 5 6 7 8 9 1 0 1 1
Lo g o f co p y n u m b er (1 0 n
)
C
t
32. Imagining
Real-Time
PCR
Measuring
Quantities
How sensitive is Real-Time PCR?
Ultimately, even a single copy can be
measured! In reality, typically about 100
copies is around the minimum amount.
One hundred copies of a 200-bp gene is
equivalent to just twenty attograms (2 x 10-17 g)
of DNA!
Copy Number vs. Ct - Standard Curve
y = -3.3192x + 39.772
R
2
= 0.9967
0
5
10
15
20
25
30
35
40
0 1 2 3 4 5 6 7 8 9 10 11
Log of copy number (10n
)
C
t
34. How do We
Measure
DNA in a
PCR
Reaction?
We use reagents that fluoresce in the
presence of amplified DNA!
Ethidium bromide and SYBR Green I
dye are two such reagents.
They bind to double-stranded DNA and
emit light when illuminated with a
specific wavelength.
SYBR Green I dye fluoresces much
more brightly than ethidium.
39. What Type
of
Instruments
are used
with Real-
Time PCR?
Real-time PCR instruments consist of THREE
main components:
1. Thermal Cycler (PCR machine)
2. Optical Module (to detect fluorescence in
the tubes during the run)
3. Computer (to translate the fluorescence
data into meaningful results)
42. What Type
of Software
is used with
Real-Time
PCR?
The real-time software converts the
fluorescent signals in each well to
meaningful data.
1. Set up PCR protocol.
2. Set up plate layout.
3. Collect data.
4. Analyze data.
1 2 3,4
44. Real-Time
PCR
Actual Data
• This is some actual data from a recent real-
time PCR run.
• Data like this can easily be generated by
preparing a dilution series of DNA.
c366939
47. Real-Time
PCR
Actual Data
• The fluorescence data collected during PCR
tells us “how much” … but there is another
type of analysis we can do that tells us
“what”!
c366939
48. 5’
5’
3’
3’
Real-Time
PCR – the
Concept of
MELT
CURVES…
• Melt curves can tell us what products are in
a reaction.
• Based on the principle that as DNA melts
(becomes single stranded), intercalating
dyes will no longer bind and fluoresce.
5’
5’
3’
3’
ID ID
ID
5’
5’
3’
3’
ID
COLD
MEDIUM
HOT
49. Real-Time
PCR – the
Concept of
MELT
CURVES…
• Melt curves can tell us what products are in
a reaction.
RFU vs T
dRFU/dT
50. Real-Time
PCR
The Concept
of MELT
CURVES
• Different amplicons will have different melt
peaks.
• Primer-Dimers will have a very different
melt peak.
Color key: Green=100X, Red=10000X, Blue=1000000X , Black=NTC.
51. Part 5:
How can we use the Crime Scene
Investigator kit to demonstrate
real-time PCR?
52. Crime Scene
Investigator
PCR Basics
Kit
An Overview
TYPICAL WORKFLOW
• Introduction to DNA profiling
• Set up PCR reactions
• Electrophorese PCR products
• Analysis and interpretation of results
53. Target
audience
• The Crime Scene Investigator PCR
Basics™ Kit is intended to be an
introduction to the polymerase chain
reaction (PCR)
• Students will have a much better
appreciation of the kit if they have
some understanding of DNA
structure and function
54. What is
DNA
profiling?
DNA profiling is the use of molecular
genetic methods to determine the
exact genotype of a DNA sample in a
way the results can basically
distinguish one human being from
another
The unique genotype of each sample
is called a DNA profile.
55. Since humans
are 99.9%
identical
where do
crime scene
investigators
look for
differences in
DNA profiles?
Crime Scene Investigators search in
areas of the genome that are unique
from individual to individual and are
“anonymous” (control no known trait or function)
The areas examined are Short
Tandem Repeats or STR’s
STR region
56. Example of
an STR
The TH01 locus contains repeats of TCAT.
CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
This example has 6 TCAT repeats.
There are more than 20 known TH01 alleles.
Each individual inherits 1 allele from each
parent.
57. Determining
genotypes for
individuals
using STRs
Ms. Smith’s TH01 locus for her two
chromosomes is given below.
What is her genotype?
MOM’S CHROMOSOME
CCC TCAT TCAT TCAT TCAT TCAT TCAT AAA
DAD’S CHROMOSOME
CCC TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT TCAT
TCAT TCAT TCAT TCAT AAA
58. To visualize
PCR products
Crime Scene
investigators
use gel
electrophoresis
(14)
(12)
(11)
(9)
(8)
(7)
(6)
(5)
(4)
(3)
(13)
(10)
TH01
alleles
Allele
ladder
Mother Father Child C Child D Child E
60. How the
Crime Scene
Kit works:
Set up PCR
reactions
1. Find the PCR tubes at your station.
Label them ‘CS’ for Crime Scene DNA,
‘A’ for Suspect A DNA, ‘B’ for Suspect B
DNA, ‘C’ for Suspect C DNA, and ‘D’ for
Suspect D DNA.
2. Keeping the tubes on ice, add 20 μl of
Master Mix + blue primers to each tube.
3. Keeping the tubes on ice, add 20 µl of
each DNA to the appropriately labeled
tube.
4. USE A FRESH TIP EACH TIME!
5. Mix and put in thermal cycler
6. Cycle ~3 hours
62. Analysis of
Results:
Who can’t be
excluded? 10
7
5
4
3
2
1
CS A B C D
genotype
5-2 7-4 5-2 7-2 10-3
AL: Allele ladder
CS: Crime Scene
A: Suspect A
B: Suspect B
C: Suspect C
D: Suspect D
AL
15
BXP007
alleles
65. Crime Scene
Investigator
PCR Basics
Kit in REAL-
TIME!
Option 1
• Introduction to DNA profiling
• Set up PCR reactions on a real-time
PCR instrument, using real-time
reagents
• Electrophorese PCR products
• Analysis and interpretation of results
Simply add this step
66. Crime Scene
Investigator
PCR Basics
Kit in REAL-
TIME!
Option 1
• View the Crime Scene PCR reactions as
they occur in real-time!
Contra Costa College, May 2006
67. Crime Scene
Investigator
PCR Basics
Kit in REAL-
TIME!
Option 1
• View the Crime Scene PCR reactions as
they occur in real-time!
Contra Costa College, May 2006
CS A B C D
68. Crime Scene
Investigator
PCR Basics
Kit in REAL-
TIME!
Option 2
• Introduction to DNA profiling
• Set up PCR reactions
• Electrophorese PCR products
• Analysis and interpretation of results
Entirely new protocol.
Use the kit components for
a complete Real-Time PCR
demonstration…
69. Crime Scene
Investigator
PCR Basics
Kit in REAL-
TIME!
Option 2
• Use the Crime Scene PCR kit as a source
for reliable target DNA and primers.
• Use a modified protocol:
– Dilute Crime Scene DNA provided with the kit
100, 10000, 1000000 fold.
– Run reactions with iQ SYBR Green Supermix on
a real-time PCR instrument.
Color key: Green=100X, Red=10000X, Blue=1000000X , Black=NTC.
70. Crime Scene
Investigator
PCR Basics
Kit in REAL-
TIME!
Option 2
• Use the Crime Scene PCR kit as a source
for reliable target DNA and primers.
• If different DNA samples are used,
interesting melt curves result because of
the different amplicons in the kit:
Color key: Green=100X, Red=10000X, Blue=1000000X , Black=NTC.
72. Crime Scene
Investigator
PCR Basics
Kit in REAL-
TIME!
Option 2
• Learning Points
– Viewing PCR reactions as they occur in real-time
•Exciting!
– Using real-time PCR to quantify DNA
•Basis of gene expression analysis, disease
diagnosis, etc.
– Measuring pipetting variation
•Run samples in duplicate for an easy test of
reproducibility
– Importance of experimental controls
•No template control and positive controls
– Melt curve analysis
•Tie concepts of the basic structure of DNA with
visible evidence that two strands can anneal and
melt.
73. Crime Scene
Investigator
Kit in Real-
Time !
•To run either of the two options,
ONLY two additional items are
needed!
•iQ SYBR Green Supermix
•A real-time PCR instrument
75. • Today we’ll use the DNA in the Crime Scene Kit to make
some dilutions for our real-time experiment!
• Each workgroup will prepare four real-time PCR
reactions:
– Unknown DNA (replicate 1)
– Unknown DNA (replicate 2)
– Unknown DNA diluted 1:100 (replicate 1)
– Unknown DNA diluted 1:100 (replicate 2)
• Each workgroup will have DNA from the Crime Scene kit
that has been diluted 1:10, 1:100, 1:1000, 1:10000, or
undiluted.
• If all goes well, you’ll be able to tell from the Ct values:
– Which unknown DNA you started with,
– How accurate your pipetting is,
– Whether your mini-dilution series demonstrates high-
efficiency PCR.
Today’s
Experiment:
An Overview
76. • Step 1:
– Make your DNA dilutions (screw-cap tubes).
– Dilute your “unknown” DNA 1:100
– 1 ul of your DNA into 99 ul of water.
• Step 2:
– Prepare your PCR tubes.
– Add 20 ul of the spiked SYBR Green Supermix
(contains 0.2 ul of Crime Scene Primers) to your four
PCR tubes.
• Step 3:
– Complete your PCR reactions.
– Add 20 ul of your DNA samples to each PCR tube.
• Two tubes undiluted, two tubes 1:100.
– Mix gently, avoiding bubbles!
• Step 4:
– Place your reactions in the real-time PCR machine.
Today’s
Experiment:
Step-By-Step
77. • Our PCR protocol will look like this:
•1. 95C for 3 min (activates Taq)
•2. 95C for 10 sec (denatures)
•3. 52C for 30 sec (extend / anneal)
•4. Plate read (captures fluorescence data)
•5. Goto Step 2 for 39 more times
Today’s
Experiment:
PCR Protocol
78. Real-Time PCR
David A. Palmer, Ph.D.
Technical Support, Bio-Rad Laboratories
Adjunct Professor, Contra Costa College
79. Webinars • Enzyme Kinetics — A Biofuels Case Study
• Real-Time PCR — What You Need To Know
and Why You Should Teach It!
• Proteins — Where DNA Takes on Form and
Function
• From plants to sequence: a six week
college biology lab course
• From singleplex to multiplex: making the
most out of your realtime experiments
explorer.bio-rad.comSupportWebinars