Name: purifying dna bands from agarose gels
Uploaded: 2021-04-07T12:11:59.000Z
Duration: 2 min 35 s
Channel: JoVE

K12 Standards

north-carolina-k-12-science-standard-course-of-study

NC K-12 Science Standard Course of Study

9-12

Biology

heredity-inheritance-and-variation-of-traits

Heredity- Inheritance and Variation of Traits

LS.Bio.8.1

DNA Agarose Gel Electrophoresis

الرحلان الكهربائي لهلام الاغاروز هو تقنية شائعة الاستخدام لفصل جزيئات الحمض النووي حسب حجمها.
يضاف Agarose - عديد السكاريد المشتق من الطحالب الحمراء - إلى مخزن مؤقت مثل Tris-acetate EDTA (TAE) أو Tris-borate EDTA (TBE) ويذوب عن طريق التسخين.
بشكل عام ، يتم إذابة 1 جرام من الاغاروز في 100 مل من المخزن المؤقت لتشكيل محلول الاغاروز بنسبة 1٪ ، على الرغم من أنه يمكن إضافة كميات مختلفة من الاغاروز اعتمادا على التطبيق.
لتلطيخ الجل ، غالبا ما تضاف صبغة فلورية ، مثل بروميد الإيثيديوم ، إلى محلول الاغاروز. تتداخل الصبغة بين قواعد الحمض النووي والتألق تحت ضوء الأشعة فوق البنفسجية.
يمكن بعد ذلك سكب المحلول في قوالب أفقية. يتم إدخال مشط في قالب الهلام لإنشاء آبار يمكن تحميل عينات الحمض النووي فيها.
يترك محلول الاغاروز دون إزعاج ليتصلب في هلام.
يتكون الجل من جزيئات الاغاروز المرتبطة بالهيدروجين التي تشكل مصفوفة مسامية تتحرك من خلالها جزيئات الحمض النووي.
يتم خلط عينات الحمض النووي مع مخزن مؤقت للتحميل وتحميلها على الجل. يحتوي مخزن التحميل المؤقت على الجلسرين ، مما يزيد من كثافة عينات الحمض النووي ويساعدها على الاستقرار في قاع الآبار.
يحتوي المخزن المؤقت أيضا على أصباغ ، مثل زيلين سيانول وبروموفينول الأزرق ، والتي تساعد في مراقبة تقدم النطاقات المهاجرة للحمض النووي.
عندما يتم تطبيق جهد كهربائي على طول الجل ، يهاجر الحمض النووي سالب الشحنة نحو القطب الموجب ، وينتقل عبر طبقات مسام الاغاروز.
تتحرك جزيئات الحمض النووي الأكبر حجما بشكل أبطأ عبر المسام من الجزيئات الأصغر ، مما يسمح بفصل الجزيئات وفقا لحجمها.
يعتمد حجم المسام على تركيز الاغاروز في المحلول. كلما زاد تركيز الاغاروز ، كان حجم المسام أصغر ، مما يساعد على فصل شظايا الحمض النووي الأقصر.
تحت ضوء الأشعة فوق البنفسجية ، تظهر جزيئات الحمض النووي الملطخة على شكل أشرطة فلورية. يمكن مقارنة موضع العصابات بشظايا الحمض النووي ذات الأحجام المعروفة ، والتي تسمى سلم الحمض النووي.
تعمل أشرطة السلم كمرجع لتحديد حجم جزيئات الحمض النووي ذات الأهمية.

<ol>
	<li>داوني ، نيكولاس. "استخراج الحمض النووي من المواد الهلامية الاغاروز." في ناقلات البلازميد الإشريكية القولونية ، ص 137-139. مطبعة هيومانا ، 2003.</li>
	<li>دي ماتوس ، جي سي بي ، إف جي إس دانتاس ، إيه كالديرا دي أراوجو ، وإم أو مورايس. "نظام الرحلان الكهربائي لهلام الاغاروز في الفصل الدراسي: الكشف عن خيط الحمض النووي يخترق تغيير طوبولوجيا البلازميد." تعليم الكيمياء الحيوية والبيولوجيا الجزيئية 32 ، العدد 4 (2004): 254-257.</li>
	<li>براون ، نورمان. "الرحلان الكهربائي لضعاف البصر: تعديل بروتوكول Lambda واستخدامه مع ضعاف البصر من المستوى A." مجلة التربية البيولوجية 29 ، العدد 3 (1995): 166-169.</li>
</ol>

الرحلان الكهربي لجل الاغاروز dna

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الفصل الكهربائي لجل الاغاروز هو تقنية مخبرية شائعة الاستخدام لفصل أجزاء الحمض النووي حسب الحجم. ومع ذلك، يمكن استخدامه أيضًا لعزل وتنقية أجزاء الحمض النووي باستخدام بروتوكول استخراج الجل.

يتبع استخراج الجل خمس خطوات رئيسية: تشغيل الفصل الكهربائي للهلام لفصل الأجزاء، وعزل العصابات الفردية، واستخراج الحمض النووي من تلك العصابات، وإزالة الصبغة والأملاح من الخليط المستخرج للحصول على الحمض النووي النقي.

في تجارب الاستنساخ، يتم الحصول على كل من أجزاء الحمض النووي المُدخلة والمتجهة بعد الهضم باستخدام نوكليازات داخلية مقيدة. يتم خلط أجزاء الحمض النووي ذات الأحجام المختلفة مع الملوثات، مثل الإنزيمات والأملاح وما إلى ذلك، والتي يمكن أن تمنع تفاعل الارتباط التالي. ولذلك يتم استخدام استخراج هلام للحصول على شظايا الحمض النووي النقي قبل الالربط.

لإعداد مادة هلامية للاستخراج، يتم استخدام محلول الاغاروز بنسبة أقل (0.7-0.8%) لضمان كفاءة الهجرة لنطاقات الحمض النووي. بالإضافة إلى ذلك، يتم استخدام قالب هلامي واسع الممشط للحصول على أشرطة DNA سميكة يسهل عزلها. بعد ذلك، يتم إجراء الفصل الكهربائي للهلام بجهد أقل لمنع تسخين الجل وتلف الحمض النووي.

بمجرد اكتمال الفصل الكهربائي للهلام، يتم التعرف على الحمض النووي الملون ببروميد الإيثيديوم عن طريق تعريض الجل للأشعة فوق البنفسجية ذات الطول الموجي الطويل لفترة قصيرة. التعرض القصير للأشعة فوق البنفسجية يمنع تلف الحمض النووي. ويتبع ذلك قطع الشريط المطلوب باستخدام شفرة حلاقة نظيفة.

تتم بعد ذلك معالجة شريحة الجل المعزولة التي تحتوي على جزء الحمض النووي محل الاهتمام من خلال إحدى مجموعات استخراج الجل المتوفرة تجاريًا. كل هذه المجموعات تتبع نفس المبدأ الأساسي. أولاً، يتم إذابة هلام الاغاروز بواسطة محلول عازل يحتوي على أملاح، مثل يوديد الصوديوم. بعد ذلك، يتم الربط الحمض النووي بعمود يحتوي على راتينج أنيوني ويتم غسله عدة مرات بمحلول كحولي مخفف لإزالة الشوائب. ثم يتم تجفيف العمود عن طريق الدوران في جهاز للطرد المركزي. يمكن الآن إزالة الحمض النووي النقي باستخدام الماء العازل أو الماء منزوع الأيونات.

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الرحلان الكهربي لجل الاغاروز DNA

الرحلان الكهربائي لهلام الاغاروز هو تقنية معملية شائعة الاستخدام لفصل شظايا الحمض النووي حسب الحجم. ومع ذلك ، يمكن استخدامه أيضا لعزل وتنقية ...

Education

JoVE Core

Molecular Biology

البيولوجيا الجزيئية

Unit 2

الوحدة الثانية

Studying DNA and RNA

دراسة الحمض النووي الريبوزي منقوص الأكسجين (DNA) والحمض النووي الريبوزي (RNA)

KEY TERMS AND CONCEPTS

المصطلحات والمفاهيم الأساسية

purifying dna bands from agarose gels

Agarose gel electrophoresis can be used to purify DNA bands after separation by size. In gel extraction, the DNA fragment of interest is cut from the gel and cleaned before later use. This is especially useful in cloning, where pure DNA is needed before ligation, the joining step that links DNA pieces together.

In cloning experiments, both the insert and vector DNA fragments are first cut with restriction endonucleases. These fragments often contain enzymes, salts, and other contaminants. Such impurities can interfere with the ligation reaction that follows, so gel extraction helps remove them before the next step.

A gel prepared for extraction uses a lower agarose concentration, about 0.7 to 0.8 percent, so DNA bands move efficiently. A wide-combed gel cast is also used to make thick bands that are easier to isolate. The gel is run at lower voltage to limit heating and reduce DNA damage.

After electrophoresis, ethidium bromide-stained DNA is viewed under long-wavelength UV light for a short time. Short UV exposure helps protect the DNA from damage. The desired band is then cut out with a clean razor blade, and the gel slice is processed with a commercial gel extraction kit.

These kits use the same basic principle. First, a salt buffer such as sodium iodide dissolves the agarose gel. The DNA then binds to a column with an anionic resin and is washed several times with a dilute alcohol solution to remove impurities. The column is spun dry in a centrifuge, and the pure DNA is finally eluted with buffer or deionized water.

Purifying DNA Bands from Agarose Gels

recombinant dna

Recombinant DNA and Gene Cloning

Recombinant DNA and gene cloning are tools used to combine DNA from different sources. Scientists use them to isolate a gene, join it to a carrier DNA molecule, and make copies of that DNA. A plasmid is one common carrier used in this process. A plasmid is a small ring of DNA found in bacteria, and it can carry the inserted gene into a host cell. Once inside the cell, the recombinant DNA can be copied as the cell grows. This approach is important in biotechnology and genetic research. It...

dna isolation

Genomic DNA Extraction Methods

Genomic DNA extraction methods separate DNA from proteins, RNA, and other cell material. The process usually follows four main steps. First, the cell structure is disrupted with mechanical or chemical methods to make a cell lysate. Then the DNA is protected from damage during processing. After that, the soluble DNA is separated from cell debris. Finally, the purified DNA is eluted, or washed out, for later use. DNA isolation can follow two main approaches: solution-based methods or solid-phase...

dna agarose gel electrophoresis

Agarose gel electrophoresis can be used to purify DNA bands after separation by size. In gel extraction, the DNA fragment of interest is cut from the gel and cleaned before later use. This is especially useful in cloning, where pure DNA is needed before ligation, the joining step that links DNA pieces together. In cloning experiments, both the insert and vector DNA fragments are first cut with restriction endonucleases. These fragments often contain enzymes, salts, and other contaminants. Such...

labeling dna probes

DNA Probe Labels for Detecting Targets

DNA probes are short DNA fragments with a reporter tag attached so they can be detected or purified. They bind to matching nucleic acid sequences by complementary base-pairing. That binding helps researchers recover or identify specific regions of DNA. Several reporter tags are commonly used to label DNA probes. These include radioisotopes, fluorophores, and small molecule binding partners such as biotin or digoxigenin. The label choice depends on the probe’s intended use and on the source of...

southern blot

Southern Blot for DNA Detection

Southern blotting is a DNA detection method used after agarose gel electrophoresis separates DNA fragments by size. A DNA ladder with fragments of known length is run beside the sample so the fragment size can be estimated. The blot adds another step when scientists need to confirm the sequence identity of the DNA. First, the target DNA is cut into smaller pieces with a restriction endonuclease and run on an agarose gel. The DNA is then denatured, which means the double strands are separated...

dna microarrays

Gene Chips for Comparing Cell Activity

DNA microarrays are gene chips used to compare gene activity in different conditions. They are high-throughput assays, which means they can analyze large amounts of data at once. They are also relatively inexpensive and can be automated, making them useful for genome-wide studies. These chips are built on glass or silica slides. Probe molecules are covalently attached to the surface through surface functionalization. In many cases, the slides are prepared by chemisorption of silanes to silica...

complementary dna

Making cDNA from RNA Templates

Complementary DNA, or cDNA, is made from an RNA template. It is a DNA copy of RNA, so it can help students study gene expression in a lab setting. cDNA is useful when scientists want to work with genes after they have been turned on in a cell. Because it is copied from RNA, it reflects which genes were active at the time the RNA was collected. This process links RNA and DNA in a simple way. It also gives students a clear view of how genetic information can be copied for analysis.

fish - fluorescent in-situ hybridization

FISH for Chromosome and Pathogen Detection

FISH is a DNA and RNA detection method used in cytogenetics and medical testing. FISH stands for fluorescence in situ hybridization. It uses labeled probes that bind to complementary DNA or RNA sequences on a chromosome or inside a cell. The method was developed in the early 1980s and became widely used because it can show specific genetic targets with high detail. In the past, probes were made by cloning or by reverse transcription of a DNA template. Today, probe oligonucleotides can also be...

pcr - polymerase chain reaction

PCR: DNA Amplification for Genetics Labs

PCR is a DNA amplification method used in genetics labs. PCR stands for polymerase chain reaction. It copies a specific DNA segment so scientists can study small amounts of genetic material. The process uses repeated heating and cooling steps. These steps let the DNA strands separate, primers bind to the target region, and a DNA polymerase build new strands. With each cycle, the target DNA can increase in amount. PCR is a core technique in molecular biology and many school-level biology...

real time rt-pcr

Real-Time RT-PCR for Gene Expression Tracking

Real-time RT-PCR tracks gene expression by measuring how much target DNA is made during PCR. It starts with mRNA, which is converted into complementary DNA, or cDNA, by an enzyme called reverse transcriptase. The cDNA is then amplified by PCR so the target sequence can be measured. This method can be done in one tube as a single-step reaction or in two separate steps. During amplification, the amount of product is followed in real time with fluorescent dyes or sequence-specific probes linked...

race - rapid amplification of cdna ends

Finding cDNA Ends with RACE PCR

Rapid Amplification of cDNA Ends, or RACE, helps scientists capture the full-length cDNA from an mRNA sequence. It works by linking a known internal region to the unknown sequence at either the 5' end or the 3' end. The method is widely used when the missing end of a gene needs to be identified. RACE uses a gene-specific primer that binds to the known cDNA region. A hybrid primer then adds a set anchor sequence to the unknown end of the cDNA. After that, PCR amplifies the DNA segment between...

sanger sequencing

How Sanger Sequencing Reads DNA Fragments

Sanger sequencing reads DNA fragments by separating pieces of different lengths and decoding the order of bases. It is a key method in biology and is often used to study a cloned DNA fragment, a gene mutation, or even much larger genomes. Fredrick Sanger and his collaborators developed the chain-termination method in 1977. The method works by producing a mixture of DNA fragments with variable sizes. These fragments are then separated by capillary gel electrophoresis, which sorts them by length.

next-generation sequencing

DNA Sequencing Methods and Uses

DNA sequencing methods can read millions of DNA fragments at the same time. Next-generation sequencing made this process much faster and much less expensive than older methods. The first human genome project cost $2.7 billion and took 15 years. It was completed in 2003 after major international cooperation among research teams and funding agencies. To prepare DNA for next-generation sequencing, pure genomic DNA is first broken into smaller pieces. These pieces are used to build a sequencing...

rna-seq

RNA-Seq Workflow and Data Analysis

RNA sequencing, or RNA-Seq, is a high-throughput method for studying the transcriptome of a cell. The transcriptome is the full set of RNA molecules in a sample. Transcriptomics helps scientists understand how genomes work, how organisms develop, and how some diseases occur. Before RNA-Seq, researchers often used microarray methods and Sanger sequencing to study RNA. Microarrays had limited coverage and depended on already knowing the genome sequence. Sanger sequencing was low-throughput,...

genome annotation and assembly

How Genome Assemblies Are Updated

Genome assembly and genome annotation are key steps in genome analysis. Genome assembly puts DNA sequencing data back in the correct order to make a close copy of the original genome. Genome annotation then identifies functional parts on that assembled genome. Assembling a genome is not simple. In human populations, gene copy number can vary, and repeated DNA sequences can make the process harder. Even so, the physical location of human genes stays constant. Bacterial genomes can be more...

restriction enzymes

DNA Cutting Sites and Bacterial Defense

Restriction enzymes are bacterial proteins that cut DNA at specific sequence sites. They recognize palindromic DNA sequences, also called inverted repeats, and cut the DNA in a sequence-specific way. These sites are often found in regions with important functions, such as the origin of replication, gene operator sites, and transcription termination signals. Bacteria protect their own DNA by methylating these same sites. Methylation means adding chemical marks that keep the restriction enzyme...

maxam-gilbert sequencing

Chemical Cleavage DNA Sequencing

Chemical cleavage DNA sequencing uses specific chemicals to cut DNA at known sites. Allan Maxam and Walter Gilbert described this method in the same year that the Sanger sequencing method was discovered. It reads the DNA sequence by breaking purified DNA into fragments and then studying the fragment pattern. The method depends on chemicals that cleave DNA at specific bases. The resulting fragments are separated by electrophoresis, which is a way to sort DNA pieces by size using an electric...

key terms and concepts

Learn how gel extraction isolates DNA bands from agarose gels, removes salts and enzymes, and prepares clean DNA for cloning and ligation.

Purifying DNA Bands from Agarose Gels | JoVE K12

molecular biology

JoVE Core Molecular Biology showcases the molecular processes of the cell through clear and concise animated video lessons. Additionally, in scientist-in-action videos, these concepts' application is demonstrated through real experiments in modern laboratories.

Tennessee Academic Standards

High School

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

Florida Department of Education

Life Science

SC.912.L.16

SC.912.L.16.12

Texas Essential Knowledge and Skills

BIO.7

BIO.7.D

What is Meiosis?

Meiosis is the process by which diploid cells divide to produce haploid daughter cells. In humans, each diploid cell contains 46 chromosomes, half from ...

Meiosis I

Meiosis II

Meiosis vs. Mitosis

Crossing Over

Crossing over is the exchange of genetic information between homologous chromosomes during prophase I of meiosis I. Genetic recombination gives rise to ...

Nondisjunction

Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate correctly and move to the opposite poles of the cells. This ...

Spermatogenesis

Spermatogenesis is the process by which haploid sperm cells are produced in the male testes. It starts with stem cells located close to the outer rim of ...

Oogenesis

In human women, oogenesis produces one mature egg cell or ovum for every precursor cell that enters meiosis. This process differs in two unique ways from ...

Fertilization

During fertilization, an egg and sperm cell fuse to create a new diploid structure. In humans, the process occurs once the egg has been released from the ...

Cleavage and Blastulation

After a large-single-celled zygote is produced via fertilization, the process of cleavage occurs while zygotes travel through the uterine tube. Cleavage ...

Gastrulation

Gastrulation establishes the three primary tissues of an embryo: the ectoderm, mesoderm, and endoderm. This developmental process relies on a series of ...

Neurulation

Neurulation is the embryological process which forms the precursors of the central nervous system and occurs after gastrulation has established the three ...

Cell Migration

Cell migration, the process by which cells move from one location to another, is essential for the proper development and viability of organisms ...

Determination

During embryogenesis, cells become progressively committed to different fates through a two-step process: specification followed by determination. ...

Use models to explain how DNA is passed from parents to offspring through the processes of meiosis and fertilization in sexual reproduction.

LS.Bio.6.1

Mutations

Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations ...

genome-size-and-the-evolution-of-new-genes

Genome Size and the Evolution of New Genes

types-of-genetic-transfer-between-organisms

Types of Genetic Transfer Between Organisms

Base-pairing and DNA Repair

The DNA Replication Fork

Lagging Strand Synthesis

The Replisome

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template ...

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the ...

Replication in Eukaryotes

In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before ...

Overview of DNA Repair

Base Excision Repair

Nucleotide Excision Repair

DNA Distortion and Damage
Cells are regularly exposed to mutagens&#8212;factors in the environment that can damage DNA and generate mutations. UV ...

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the ...

Fixing Double-strand Breaks

Homologous Recombination

Gene Conversion

Restarting Stalled Replication Forks

Construct an explanation to summarize how inheritable genetic variations may result from: new genetic combinations in meiosis, mutations during replication, or mutations caused by environmental factors.

LS.Bio.6.2

An Overview of Genetic Analysis

An organism&#8217;s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its ...

Genetic Crosses

To dissect genetic processes or create organisms with novel suites of traits, scientists can perform genetic crosses, or the purposeful mating of two ...

Punnett Squares

Monohybrid Crosses

Dihybrid Crosses

Trihybrid Crosses

Trihybrid Crosses
Some of Mendel&#8217;s crosses examined three pairs of contrasting characteristics. Such a cross is called a trihybrid cross. A ...

Law of Independent Assortment

Chi-square Analysis

The chi-square test is a statistical hypothesis test. It is used to check whether there is a significant difference between an expected value and an ...

Pedigree Analysis

Multiple Allele Traits

Incomplete Dominance

Gregor Mendel&#39;s work (1822 - 1884) was primarily focused on pea plants. Through his initial experiments, he determined that every gene in a diploid ...

Lethal Alleles

Agouti: A Lethal Allele
Lucien Cu&#233;not discovered lethal alleles in 1905 while studying the inheritance of coat color in mice. The agouti gene is ...

Polygenic Traits

background-and-environment-affect-phenotype

Background and Environment Affect Phenotype

Although the genetic makeup of an organism plays a major role in determining the phenotype, there are also several environmental factors, such as ...

Use mathematics and computational thinking to predict the variation and distribution of expressed traits based on: Mendelian inheritance, co-dominance, incomplete dominance, multiple alleles, and sex-linked inheritance.

LS.Bio.7.1

Overview

A pedigree is a diagram displaying a family&#x2019;s history of a trait. Analyzing pedigrees can reveal (1) whether a trait is dominant or ...

Probability Laws

Overview

The probability of inheriting a trait can be calculated using the sum and product rules. The sum rule is used to calculate the probability of ...

The Concept of Multiple Allelism

Multiple allelism describes genes that exist in three or more allelic forms. Although diploid organisms, like humans, ...

Analyze and interpret data to explain how polygenic traits result in a wide range of phenotypes.

LS.Bio.7.2

Pleiotropy

Pleiotropy is the phenomenon in which a single gene impacts multiple, seemingly unrelated phenotypic traits. For example, defects in the SOX10 gene cause ...

Nature and Nurture

Many human characteristics, like height, are shaped by both nature&mdash;in other words, by our genes&mdash;and by nurture, or our environment. For ...

Construct an explanation to summarize how traits result from interactions of genetic factors (multiple genes and/or alleles) and environmental factors.

LS.Bio.7.3

PCR: The Polymerase Chain Reaction

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling &#8211; cyles of temperature changes at fixed time ...

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further ...

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called ...

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify ...

dna-isolation-and-restriction-enzyme-analysis

DNA Isolation and Restriction Enzyme Analysis

The revelation of DNA as the hereditary molecule in all organisms has led to enormous scientific and medical breakthroughs and significantly enhanced our ...

Preparation of Solutions and Materials
ExpandNOTE: For this lab to run smoothly, it is recommended to not exceed 12 samples per laboratory class.
First ...

DNA Isolation
ExpandNOTE: In this experiment you will perform DNA isolation under two experimental conditions: one using a buffer containing the detergent ...

Analyze and interpret data to compare DNA samples.

Understand genetic mechanisms for variation.

Purifying DNA Bands from Agarose Gels

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