The document outlines the steps and parameters for performing PCR amplification, including denaturation, annealing, and elongation temperatures and times. It discusses optimizing PCR by calculating primer melting temperatures and adjusting extension times based on expected product size. The document also describes techniques to improve specificity, such as hot start PCR and touchdown PCR. Finally, it assigns students to research a paper using PCR and describe the application in about 200 words.
what is sandwich elisa, introduction to elisa, its type and main focus on sandwich elisa, , its process and advantages along with the disadvantages, its applications
Antibody Based Techniques Masterclass by ProteintechProteintech Group
Tips to optimize your antibody based lab techniques, covering common antibody applications including Western blot, Immunohistochemistry (IHC), Immunoprecipotation (IP) and Immunofluorescence (IF).
Proteintech technical workshops are coordinated by Dr Szczesna (Proteintech's technical expert) and cover a range of topics including step-by-step protocol optimization, FAQS and troubleshooting tips.
what is sandwich elisa, introduction to elisa, its type and main focus on sandwich elisa, , its process and advantages along with the disadvantages, its applications
Antibody Based Techniques Masterclass by ProteintechProteintech Group
Tips to optimize your antibody based lab techniques, covering common antibody applications including Western blot, Immunohistochemistry (IHC), Immunoprecipotation (IP) and Immunofluorescence (IF).
Proteintech technical workshops are coordinated by Dr Szczesna (Proteintech's technical expert) and cover a range of topics including step-by-step protocol optimization, FAQS and troubleshooting tips.
Polymerase chain reaction (abbreviated PCR) is a laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. PCR involves using short synthetic DNA fragments called primers to select a segment of the genome to be amplified, and then multiple rounds of DNA synthesis to amplify that segment. This slides introduces pcr importances ,uses and steps of pcr.
MOLECULAR TOOLS IN DIAGNOSIS AND CHARACTERIZATION OF INFECTIOUS DISEASES tawheedshafi
The future of the molecular diagnostics of infectious diseases will undoubtedly be focused on a marked increase in the amount of information detected with remarkably simplified, rapid platforms that will need complex software analysis to resolve the data for use in clinical decision-making.
All about polymerase chain reaction. detailed description and explanation of instrumention, procedure, advantages, disadvantages. Also types of RtPcr..
graphical representation. explained with appropriate figrues.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
Basic Molecular Biology:
Molecular biology is the branch of biology that focuses on understanding the fundamental processes and mechanisms underlying life at the molecular level. It involves the study of biological molecules such as DNA, RNA, and proteins, and how they interact to regulate various cellular processes. Molecular biology techniques enable scientists to investigate genetic information, gene expression, and the structure and function of macromolecules.
Polymerase Chain Reaction (PCR):
Polymerase Chain Reaction (PCR) is a powerful molecular biology technique used to amplify and replicate a specific segment of DNA in a laboratory setting. PCR allows scientists to make millions of copies of a target DNA sequence in a short period. It consists of repeated cycles of denaturation (separation of DNA strands), annealing (binding of short DNA primers to the target sequence), and extension (synthesis of new DNA strands using a heat-stable DNA polymerase enzyme). PCR has diverse applications, including DNA sequencing, genetic testing, forensics, and the study of gene expression.
Reverse Transcription Polymerase Chain Reaction (RT-PCR):
Reverse Transcription Polymerase Chain Reaction (RT-PCR) is a variation of the standard PCR technique that is specifically used to amplify RNA molecules. It involves a two-step process. First, the RNA is reverse transcribed into complementary DNA (cDNA) using the enzyme reverse transcriptase. Then, the cDNA is amplified using standard PCR. RT-PCR is essential for studying gene expression, viral RNA detection (e.g., for diagnosing diseases like COVID-19), and a range of other applications where RNA analysis is crucial.
June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
Acetabularia Information For Class 9 .docxvaibhavrinwa19
Acetabularia acetabulum is a single-celled green alga that in its vegetative state is morphologically differentiated into a basal rhizoid and an axially elongated stalk, which bears whorls of branching hairs. The single diploid nucleus resides in the rhizoid.
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
Francesca Gottschalk - How can education support child empowerment.pptxEduSkills OECD
Francesca Gottschalk from the OECD’s Centre for Educational Research and Innovation presents at the Ask an Expert Webinar: How can education support child empowerment?
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
Synthetic Fiber Construction in lab .pptxPavel ( NSTU)
Synthetic fiber production is a fascinating and complex field that blends chemistry, engineering, and environmental science. By understanding these aspects, students can gain a comprehensive view of synthetic fiber production, its impact on society and the environment, and the potential for future innovations. Synthetic fibers play a crucial role in modern society, impacting various aspects of daily life, industry, and the environment. ynthetic fibers are integral to modern life, offering a range of benefits from cost-effectiveness and versatility to innovative applications and performance characteristics. While they pose environmental challenges, ongoing research and development aim to create more sustainable and eco-friendly alternatives. Understanding the importance of synthetic fibers helps in appreciating their role in the economy, industry, and daily life, while also emphasizing the need for sustainable practices and innovation.
1. Dr. Michael Bulger, MRBX 1-11108
Dr. Yi-Tao Yu
Dr. Peter Gibbs
Series of lectures/short talks
3 write ups; schedule flexible depending on progress and other
commitments.
3 quizzes; READ AHEAD. One assignment.
ASK Questions
2. EXPERIMENTAL OVERVIEW
PCR a DNA fragment using a plasmid template
Identify and purify the PCR product
Digest the product with suitable restriction enzymes
Ligate the product into a suitably digested cloning vector
Transform E. coli
Identify colonies containing the insert
Confirm the clone by miniprep of DNA and restriction digest
Perform large-scale DNA preparation
Sequence the chosen subclone
Analyze sequence by database searching/bioinformatics
3. EXPERIMENTAL OVERVIEW
PCR a DNA fragment using a plasmid template
Identify and purify the PCR product
Digest the product with suitable restriction enzymes
Ligate the product into a suitably digested cloning vector
Transform E. coli
Identify colonies containing the insert
Confirm the clone by miniprep of DNA and restriction digest
Perform large-scale DNA preparation
Sequence the chosen subclone
Analyze sequence by database searching/bioinformatics
25. The PCR Amplification Plateau
Given that the individual components of a PCR reaction are not consumed and therefore
do not become rate limiting, what is the cause of the amplification plateau ?
• Reagents unstable at high temps; Taq DNA polymerase has a finite half-life,
• Substrate excess; more DNA product present than remaining Taq can replicate in a given time,
• Incomplete template strand denaturation at high concentrations,
• Strand reannealing of specific product at high concentration prevents primer annealing,
• Competition for reactants by non-specific products-especially shorter products and primer dimers.
26.
27. Hot Start PCR
During sample preparation at room temperature complexes of nonspecific primer-template or
primer-primer may be generated. With HotStart a key component, such as primers, polymerase,
Mg++, or dNTPs,necessary for amplification is withheld from the reaction mix until the
reaction reaches a temperature above the optimal annealing temperature of the primers.
In this way competing side reactions are minimized and a more specific amplification can occur.
• Add Taq manually to every tube when reaction reaches 94°C
• "Hot Wax" beads which contain MgCl2 emulsifed in paraffin beads.
• TaqBead, 1.25 ul/bead, facilitates hot start PCR by releasing Taq at 60°C
• Anti-Taq antibody; A heat labile antibody maintains Taq in an inactive state until ~60°C
28. Touchdown PCR
Touchdown PCR is a technique devised to minimize the impact of nonspecific annealing of primers to
template during PCR. It takes advantage of the exponential amplification provided by PCR to favor the
production of specific products.
In this technique, the initial annealing temperature is set just below the calculated Tm for the primers.
In a typical protocol, the annealing temperature is reduced by 1°C every 2 cycles (although there are many
variations).
Only the most specific primer-template interactions will take place at the highest annealing temperatures
(likely to be at the sequence of interest), and so in later rounds, when the annealing temperature is lower,
the specific products will “swamp out” the nonspecific products, even though all will be amplified.
29. Quantitative Real-Time PCR (qrt-PCR)
--qrt-PCR is a variation on PCR in which product amplification is measured quantitatively during cycling.
--This is accomplished by detection of a fluorescent signal that is produced proportionally with PCR
product.
Example: SYBR-green
DNA intercalating dye, but only fluoresces when intercalated in double-stranded DNA.
Thus, as cycling proceeds and the product is amplified, fluorescence can be measured during
the annealing step.
30. Quantitative Real-Time PCR (qrt-PCR)
SYBR-green not specific - will detect all products, not just specific products.
More specificity can be achieved using probe-based (i.e. “Taqman”) approaches.
31. ASSIGNMENT
Find ANY paper that describes an application of PCR or uses PCR
as a method for accomplishing the author’s experimental goals.
In ~200 words describe the use of PCR in their particular
experiment; the template, the primers (sequence, Tm [calculate it
yourself if not stated] and other characteristics, the size of the
products, how they were analyzed and the downstream application
of their PCR products.
Hand in next week i.e. 7 days time.
32. A. Determine thermal cycling parameters.!
!
1. Using the following guidelines, determine the thermal cycling parameters of the reaction and program the!
thermal cycler. (Fill in the annealing temperature and extension times according to primer and product!
considerations; see the pre-lab handout for help). Run the program for 30 cycles.!
!
0.5
94
Denature ___ min at ___°C !
!
0.5
50
Anneal ___ min at _____ °C:!
!
If primer GC content is <=50%, anneal at 55°C; if >50%, anneal at 60°C. Also, calculate the Tms of the!
two primers using the '2+4' rule to assess the validity of the above guideline.!
!
52
56
Primer 1; ___°C; Primer 2; ___°C.!
!
1.5
72
Extend _____ min at ___°C:!
!
If product length is <=500 nucleotides, extend 1 min; if >500 nucleotides, extend 3 min.!
!
Your product is anticipated to be ~500bp.!
!
!
Lengthen the time for the last extension step of the last cycle to 7 min, to ensure that all the PCR products!
!are full-length.!
