Cloning : DNA preparation
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DNA preparation and cloning techniques involve several steps: 1. Restriction enzyme digestion cuts DNA at specific recognition sites, producing blunt or overhanging ends. This allows directional cloning but has sequence constraints. 2. TA cloning avoids restriction enzymes by relying on A/T base pairing, providing high efficiency but limited vector choices and lack of sequence control. 3. Seamless cloning uses DNA polymerase and ligase to join overlapping fragments without extra sequences. This provides exquisite gene assembly control but is more costly than traditional methods. 4. Ligation independent cloning uses exonuclease and complementarity between vector and insert overhangs for fast, versatile cloning without ligase. However, some sequence modifications are not possible
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TA cloning is a subcloning technique that relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize and ligate together without using restriction enzymes. PCR products are amplified with Taq DNA polymerase, which adds an adenine to the 3' end. These inserts are cloned into vectors that are linearized and given complementary 3' thymine overhangs. The process is simpler and faster than traditional cloning as it does not require restriction enzymes, with commercial kits expediting the workflow, though the gene has a 50% chance of inserting in the reverse direction.
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TA cloning is a cloning technique that avoids restriction enzymes. It relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize when ligated together. The technique involves using a PCR product amplified with Taq polymerase, which leaves 3' adenine overhangs, and a linearized vector with 3' thymine overhangs. The complementary overhangs allow the PCR product to ligate directly into the vector. Examples of vectors designed for TA cloning, such as pLUG vectors, were also discussed. TOPO TA cloning uses topoisomerase enzyme to ligate PCR products to vectors. Ligation independent cloning uses annealing of single-stranded overhangs of at least 12 bases
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TA cloning is a subcloning technique that relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize and ligate together without using restriction enzymes. PCR products are amplified with Taq DNA polymerase, which adds an adenine to the 3' end. These inserts are cloned into vectors that are linearized and given complementary 3' thymine overhangs. The process is simpler and faster than traditional cloning as it does not require restriction enzymes, with commercial kits expediting the workflow, though the gene has a 50% chance of inserting in the reverse direction.
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PCR is a technique that amplifies a specific region of DNA using oligonucleotide primers and DNA polymerase. The primers are complementary to the outer regions of the target DNA sequence. During PCR, the DNA is denatured and the primers anneal to the single-stranded DNA. DNA polymerase then synthesizes new DNA strands that are complementary to the parent strands. This results in exponential amplification of the target DNA sequence.
AFLP, RFLP & RAPD
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
DNA manipulation Enzymes 2.pdf
The document discusses enzymes used for DNA manipulation, which fall into four main categories: DNA polymerases, nucleases, ligases, and end-modification enzymes. DNA polymerases are template dependent and include DNA polymerase 1. Nucleases include exonucleases like Bal31 and endonucleases like DNase I. Ligases involve T4 DNA ligase. End-modification enzymes include alkaline phosphatase. Restriction endonucleases recognize specific DNA sequences and have various applications in genetic engineering.
Molecular marker technology in studies on plant genetic diversity
A molecular marker is a molecule contained within a sample taken from an organism (biological markers) or other matter. It can be used to reveal certain characteristics about the respective source. DNA, for example, is a molecular marker containing information about genetic disorders, genealogy and the evolutionary history of life. Specific regions of the DNA (genetic markers) are used to diagnose the autosomal recessive genetic disorder cystic fibrosis, taxonomic affinity (phylogenetics) and identity (DNA Barcoding). Further, life forms are known to shed unique chemicals, including DNA, into the environment as evidence of their presence in a particular location.Other biological markers, like proteins, are used in diagnostic tests for complex neurodegenerative disorders, such as Alzheimer's disease. Non-biological molecular markers are also used, for example, in environmental studies.
Application of Genetic Analyzer in AFLP Technique
The presentation introducing the following points:
•Genetic markers.
•Restriction Fragment Length Polymorphism (RFLP).
•Amplified Fragment Length Polymorphism (AFLP).
•Using Genetic Analyzer in AFLP.
•Troubleshooting.
© FAO: http://www.fao.org
PCR and primer design techniques
The document discusses various techniques and applications of polymerase chain reaction (PCR). It describes the invention of PCR in 1982 and some key adaptations such as real-time PCR, which allows for quantitative analysis of DNA amplification in real time using fluorescent probes. Different types of PCR are also summarized, including reverse transcription PCR, nested PCR, multiplex PCR, and touchdown PCR. Components of real-time PCR and steps in the PCR process are outlined.
Pcr
This document provides information on three molecular biology techniques: polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and random amplified polymorphic DNA (RAPD). It describes the history, principles, requirements, procedures, applications, and variations of PCR. It also briefly discusses the principles, procedures, advantages, and limitations of RFLP and RAPD techniques.
Dna cloning by utkarsh
DNA cloning is a technique used to reproduce DNA fragments. It involves inserting a DNA fragment into a vector, which is then used to transform host cells. The key steps are digesting the DNA and vector with restriction enzymes, ligating the DNA fragment into the vector, transforming host cells with the vector, and selecting for cells containing the vector through growth on antibiotic-containing media. This allows unlimited amplification of the DNA fragment for study, manipulation, and protein expression.
PROKAYOTIC TRANSCRIPTION.pptx
Transcription in prokaryotes involves RNA polymerase copying a portion of the DNA genome into RNA. It occurs in three main steps: initiation, elongation, and termination. Initiation begins with RNA polymerase binding to promoter sequences on DNA to form a closed complex, then an open complex. Elongation entails RNA polymerase continuing to synthesize RNA down the DNA template. Termination can occur via either rho-independent or rho-dependent mechanisms, stopping further RNA production.
Pcr
The document summarizes several molecular biology techniques used to analyze DNA, including PCR, RFLP, DGGE, FISH, and clone libraries. PCR is used to amplify a targeted region of DNA using primers. RFLP and DGGE separate DNA fragments of different lengths after restriction enzyme digestion or denaturation to identify sequences. FISH uses fluorescent probes to directly image specific organisms in samples. Clone libraries create collections of DNA fragments for sequencing.
Various tools used in genetic engineering
genetic engineering is the most successful technique under biotechnology .Here review on various tool of genetic engineering is discussed. #knowmore.
C dna library
A DNA library prepared from m RNA and useful to explore new gene and other tissues specific gene expression
Pcr
The document discusses several types and applications of polymerase chain reaction (PCR). It begins by explaining the basic three-step cycling process of PCR: denaturation, annealing of primers, and extension. It then describes several variations of PCR including inverse PCR, anchored PCR, asymmetric PCR, real-time PCR (RT-PCR), and PCR for site-directed mutagenesis. Inverse PCR is used to amplify unknown flanking genomic regions, while anchored and asymmetric PCR are used to generate single-stranded DNA products for downstream applications like sequencing. RT-PCR amplifies RNA sequences by first generating cDNA. PCR mutagenesis introduces mutations through altered primer sequences.
Dna footprinting
DNA footprinting is a technique to study how proteins interact with DNA. It involves treating DNA with nucleases or chemicals that cut DNA, but are blocked from cutting where a protein is bound. This leaves a "footprint" of protected DNA that reveals the protein's binding site. The technique was developed in 1978 and can detect even transient protein-DNA interactions to help understand transcriptional control within cells.
Lectut btn-202-ppt-l29. applications of pcr-i (1)
PCR can be used for a variety of applications including isolating and cloning genes, cDNA synthesis, mutagenesis, molecular markers, analyzing ancient DNA, medical diagnostics, and forensics. Molecular biologists use PCR to identify specific genes and make multiple copies. Forensic technicians use PCR to help identify suspects and victims based on DNA amplification patterns. PCR has impacted many areas of life and promises further exciting discoveries.
Molecular markers- RFLP, RAPD, AFLP, SNP etc.
Molecular markers are identifiable DNA sequences used to locate genes associated with specific traits or genetic conditions.
A molecular marker is a specific gene fragment present at a specific position called ‘locus’ (pleural loci) in the genome of a cell.
In the pool of unknown DNA or in a whole chromosome, these molecular markers help in identification of particular sequence of DNA at particular location.
Next Generation Sequencing
Sequencing is one of the major technological advancement that has taken shape in the last two or three decade. Starting from Sanger and Maxam-Gilbert sequencing methods to the latest high-throughput methods, sequencing technologies has changed the the landscape of biological sciences.
This slide takes a look a the major sequencing methods over time.
Note: Several images included here have been sourced from GOOGLE IMAGES. The content has been extracted from several SCIENTIFIC PAPERS and WEBSITES.
PLEASE DO CONTACT THE AUTHOR DIRECTLY IF ANY COPYRIGHT ISSUE ARISES.
Recombinant dna technology and DNA sequencing
title: recombinant DNA technology and DNA sequencing
this lect will cover the pcr, isolation of DNA, detection of DNA and DNA manipulation joining DNA together. this is very important and it is required in research of every field especially medical related field.
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DNA Fingerprinting (AFLP, RFLP, RAPD) & Its advntages and application
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在线办理(salfor毕业证书)索尔福德大学毕业证毕业完成信一模一样
学校原件一模一样【微信：741003700 】《(salfor毕业证书)索尔福德大学毕业证》【微信：741003700 】学位证，留信认证（真实可查，永久存档）原件一模一样纸张工艺/offer、雅思、外壳等材料/诚信可靠,可直接看成品样本，帮您解决无法毕业带来的各种难题！外壳，原版制作，诚信可靠，可直接看成品样本。行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备。十五年致力于帮助留学生解决难题，包您满意。
本公司拥有海外各大学样板无数，能完美还原。
1:1完美还原海外各大学毕业材料上的工艺：水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠。文字图案浮雕、激光镭射、紫外荧光、温感、复印防伪等防伪工艺。材料咨询办理、认证咨询办理请加学历顾问Q/微741003700
【主营项目】
一.毕业证【q微741003700】成绩单、使馆认证、教育部认证、雅思托福成绩单、学生卡等！
二.真实使馆公证(即留学回国人员证明,不成功不收费)
三.真实教育部学历学位认证（教育部存档！教育部留服网站永久可查）
四.办理各国各大学文凭(一对一专业服务,可全程监控跟踪进度)
如果您处于以下几种情况：
◇在校期间，因各种原因未能顺利毕业……拿不到官方毕业证【q/微741003700】
◇面对父母的压力，希望尽快拿到；
◇不清楚认证流程以及材料该如何准备；
◇回国时间很长，忘记办理；
◇回国马上就要找工作，办给用人单位看；
◇企事业单位必须要求办理的
◇需要报考公务员、购买免税车、落转户口
◇申请留学生创业基金
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
ESA/ACT Science Coffee: Diego Blas - Gravitational wave detection with orbita...
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Speaker: Diego Blas (IFAE/ICREA)
Title: Gravitational wave detection with orbital motion of Moon and artificial
Abstract:
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The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
Bob Reedy - Nitrate in Texas Groundwater.pdf
Presented at June 6-7 Texas Alliance of Groundwater Districts Business Meeting
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Evaporation step counter work. I have done a physical experiment.
(Work in progress.)
快速办理(UAM毕业证书)马德里自治大学毕业证学位证一模一样
学校原件一模一样【微信：741003700 】《(UAM毕业证书)马德里自治大学毕业证学位证》【微信：741003700 】学位证，留信认证（真实可查，永久存档）原件一模一样纸张工艺/offer、雅思、外壳等材料/诚信可靠,可直接看成品样本，帮您解决无法毕业带来的各种难题！外壳，原版制作，诚信可靠，可直接看成品样本。行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备。十五年致力于帮助留学生解决难题，包您满意。
本公司拥有海外各大学样板无数，能完美还原。
1:1完美还原海外各大学毕业材料上的工艺：水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠。文字图案浮雕、激光镭射、紫外荧光、温感、复印防伪等防伪工艺。材料咨询办理、认证咨询办理请加学历顾问Q/微741003700
【主营项目】
一.毕业证【q微741003700】成绩单、使馆认证、教育部认证、雅思托福成绩单、学生卡等！
二.真实使馆公证(即留学回国人员证明,不成功不收费)
三.真实教育部学历学位认证（教育部存档！教育部留服网站永久可查）
四.办理各国各大学文凭(一对一专业服务,可全程监控跟踪进度)
如果您处于以下几种情况：
◇在校期间，因各种原因未能顺利毕业……拿不到官方毕业证【q/微741003700】
◇面对父母的压力，希望尽快拿到；
◇不清楚认证流程以及材料该如何准备；
◇回国时间很长，忘记办理；
◇回国马上就要找工作，办给用人单位看；
◇企事业单位必须要求办理的
◇需要报考公务员、购买免税车、落转户口
◇申请留学生创业基金
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
Micronuclei test.M.sc.zoology.fisheries.
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The binding of cosmological structures by massless topological defects
Assuming spherical symmetry and weak field, it is shown that if one solves the Poisson equation or the Einstein field
equations sourced by a topological defect, i.e. a singularity of a very specific form, the result is a localized gravitational
field capable of driving flat rotation (i.e. Keplerian circular orbits at a constant speed for all radii) of test masses on a thin
spherical shell without any underlying mass. Moreover, a large-scale structure which exploits this solution by assembling
concentrically a number of such topological defects can establish a flat stellar or galactic rotation curve, and can also deflect
light in the same manner as an equipotential (isothermal) sphere. Thus, the need for dark matter or modified gravity theory is
mitigated, at least in part.
Immersive Learning That Works: Research Grounding and Paths Forward
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
11.1 Role of physical biological in deterioration of grains.pdf
Storagedeteriorationisanyformoflossinquantityandqualityofbio-materials.
Themajorcausesofdeteriorationinstorage
•Physical
•Biological
•Mechanical
•Chemical
Storageonlypreservesquality.Itneverimprovesquality.
Itisadvisabletostartstoragewithqualityfoodproduct.Productwithinitialpoorqualityquicklydepreciates
(June 12, 2024) Webinar: Development of PET theranostics targeting the molecu...
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Targeting Hsp90 and its pathogen Orthologs with Tethered Inhibitors as a Diagnostic and Therapeutic Strategy for cancer and infectious diseases with Dr. Timothy Haystead.The cost of acquiring information by natural selection
This is a short talk that I gave at the Banff International Research Station workshop on Modeling and Theory in Population Biology. The idea is to try to understand how the burden of natural selection relates to the amount of information that selection puts into the genome.
It's based on the first part of this research paper:
The cost of information acquisition by natural selection
Ryan Seamus McGee, Olivia Kosterlitz, Artem Kaznatcheev, Benjamin Kerr, Carl T. Bergstrom
bioRxiv 2022.07.02.498577; doi: https://doi.org/10.1101/2022.07.02.498577
8.Isolation of pure cultures and preservation of cultures.pdf
Isolation of pure culture, its various method.
Equivariant neural networks and representation theory
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
Recently uploaded (20)
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Cloning : DNA preparation
- 1. CLONING : DNA PREPARATION Phatareeya Laochinchat
- 4. Restriction enzyme (RE) digestion • RE is an enzyme that cuts DNA at or near specific recognition nucleotide sequences known as restriction sites. • RE function by cutting double stranded DNA at specific 4 to 8 base pair. • The products of DNA cleavage are either blunt ended or contain 5’ or 3’ overhangs.
- 9. Advantages/Disadvantages Advantages Disadvantages Low cost Possible sequence constraints due to presence and/or translation of restriction site Versatile Many different vector choices Directional cloning can be easily done
- 10. TA (PCR Cloning) • TA cloning is a subcloning technique that avoids the use of restriction enzymes and is easier and quicker than traditional subcloning. • The technique relies on the ability of adenine (A) and thymine (T) on different DNA fragments to hybridize.
- 11. • PCR products are usually amplified using Taq DNA polymerase which preferentially adds an A to the 3' end of the product. • PCR amplified inserts are cloned into linearized vectors that have complementary 3' T overhangs. TA (PCR Cloning)
- 13. Advantages Disadvantages High efficiency, with dedicated vectors Limited vector choices Amenable to high throughput Higher cost Lack of sequence control at junction Multi-fragment cloning is not straight forward Advantages/Disadvantages
- 14. Seamless Cloning (Gene Assembly) • Gibson Assembly enables multiple, overlapping DNA fragments to be joined in a single-tube isothermal reaction, with no additional sequence added (scar-less).
- 17. Advantages Disadvantages Sequence-dependent Cost, relative to traditional methods High cloning efficiency PCR primers for vector and insert must be designed/ordered together Efficiency assembly of multiple fragment Exquisite control of higher- order gene assembly Advantages/Disadvantages
- 18. LIC (Ligation Independent Cloning) • Ligation Independent Cloning (LIC) is a technique developed in the early 1990s as an alternative to restriction enzyme/ligase cloning. • Inserts are usually PCR amplified and vectors are made linear either by restriction enzyme digestion or by PCR. • This creative technique uses the 3’ → 5’ exonuclease activity of T4 DNA Polymerase to create overhangs with complementarity between the vector and insert.
- 20. Preparation of vector DNA
- 21. Preparation of the insert
- 22. Annealing of the insert and the LIC vector
- 23. Advantages Disadvantages Low cost Some types of sequence modifications not possible Fast Many different vector choices Advantages/Disadvantages
- 24. CLONING : DNA END MODIFICATIONS
- 25. Dephosphorylation Blunting • Dephosphorylation is a common step in traditional cloning to ensure the vector does not re-circularize during ligation. • Fragments generated by restriction digestion have a 5'- phosphate even after they have been treated with T4 DNA Polymerase for blunting. • Use Alkaline Phosphatase to remove the 5´ phosphate AP
- 26. AP
- 27. • Phosphorylation of insert DNA that lacks terminal 5' phosphates, such as PCR products and fragments with synthetic linkers. • T4 polynucleotide kinase can be used; this enzyme catalyzes the transfer of phosphate from ATP to the 5' terminus of dephosphorylated DNA or RNA Phosphorylation of Insert DNA
- 29. Blunting/End-repair • Blunt ends are always compatible with each other, because there are no H-bonds being formed that would define compatibility or incompatibility. • Cohesive ends
- 31. Ligation • Ligation is the process of joining two pieces of DNA from different sources together through the formation of a covalent bond. • DNA ligase is the enzyme used to catalyze this reaction. • DNA ligation requires ATP.
Editor's Notes
- Cloning
- เอนไซม์ตัดจำเพาะหรือ restriction enzyme เป็นเอนไซม์ตัดดีเอ็นเอในตำแหน่งที่มีลำดับเบสจำเพาะ ทำให้ได้ปลายดีเอ็นเอ 2 แบบ คือ ปลายเหนียว (sticky end) ซึ่งเป็นปลายที่มีสายดีเอ็นเอสายหนึ่งยื่นออกมา และปลายเรียบ (blunt end) ซึ่งเป็นปลายที่ไม่มีการยื่นออกมาของสายดีเอ็นเอ เมื่อตัดดีเอ็นเอ 2 ตัวอย่างด้วยเอนไซม์ชนิดเดียวกัน ทำให้ดีเอ็นเอทั้งสองนั้นเชื่อมต่อกันได้พอด