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TA Cloning (PCR cloning)
 
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This lecture explains about TA cloning process in details. TA cloning is also known as PCR cloning. PCR amplification of the target DNA using T and A base sequence is achieved and then the amplified target products can be easily cloned without the use of any restriction endonuclease enzyme. This DNA cloning technique is a hasslefree technique to clone target DNA using only the DNA of interest, the DNA vector and the DNA Ligase enzyme. Once this video lecture thoroughly to understand about the TA DNA cloning mechanism in details. For more information, log on to- http://www.shomusbiology.com/ Get Shomu's Biology DVD set here- http://www.shomusbiology.com/dvd-store/ Download the study materials here- http://shomusbiology.com/bio-materials.html Remember Shomu’s Biology is created to spread the knowledge of life science and biology by sharing all this free biology lectures video and animation presented by Suman Bhattacharjee in YouTube. All these tutorials are brought to you for free. Please subscribe to our channel so that we can grow together. You can check for any of the following services from Shomu’s Biology- Buy Shomu’s Biology lecture DVD set- www.shomusbiology.com/dvd-store Shomu’s Biology assignment services – www.shomusbiology.com/assignment -help Join Online coaching for CSIR NET exam – www.shomusbiology.com/net-coaching We are social. Find us on different sites here- Our Website – www.shomusbiology.com Facebook page- https://www.facebook.com/ShomusBiology/ Twitter - https://twitter.com/shomusbiology SlideShare- www.slideshare.net/shomusbiology Google plus- https://plus.google.com/113648584982732129198 LinkedIn - https://www.linkedin.com/in/suman-bhattacharjee-2a051661 Youtube- https://www.youtube.com/user/TheFunsuman Thank you for watching
Views: 21247 Shomu's Biology
Overview of PCR Cloning
 
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PCR Cloning is an easy and reliable cloning method utilizing DNA amplification to generate the amplicon. Learn more at https://www.neb.com/applications/cloning-and-synthetic-biology/pcr-cloning
Views: 36971 New England Biolabs
Agarose Gel Electrophoresis
 
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For more information, visit http://www.bio-rad.com/yt/idea. This video demonstrates how to load and run DNA samples on an agarose gel. Basic information about the charge of DNA and how it will run in an horizontal electrophoresis cell is explained. http://www.bio-rad.com/evportal/destination/commerce/product_detail?catID=M5H6PI15&WT.mc_id=yt-lse-ww-biotech-20121012-vq759wKCCUQ We Are Bio-Rad Explorer. Our Mission: Bio-Rad’s Explorer program provides easy access to engaging hands-on science learning experiences that spark interest in science and its influence on the world. To do this we: Provide high quality, relevant, learning experiences based on real world science Empower educators with skills and confidence to deliver engaging, memorable lessons Connect with Bio-Rad Explorer Online: Website: http://www.bio-rad.com/en-us/education Twitter: https://twitter.com/BioRadEducation Pinterest: https://www.pinterest.com/bioradexplorer/ LinkedIn: https://www.linkedin.com/company/1613226/ Facebook: https://www.facebook.com/biorad/ Instagram: @BioRadLabs Snapchat: @BioRadLabs
Views: 437812 Bio-Rad Laboratories
Plasmid as a cloning vector
 
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This dna cloning lecture explains use of plasmid as a cloning vector. http://shomusbiology.weebly.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.[1] The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use. There are many types of cloning vectors, but the most commonly used ones are genetically engineered plasmids. Cloning is generally first performed using Escherichia coli, and cloning vectors in E. coli include plasmids, bacteriophages (such as phage λ), cosmids, and bacterial artificial chromosomes (BACs). Some DNA however cannot be stably maintained in E. coli, for example very large DNA fragment, and other organisms such as yeast may be used. Cloning vectors in yeast include yeast artificial chromosomes (YACs). Plasmid is an autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the most commonly used. Most general plasmids may be used to clone DNA insert of up to 15 kb in size. One of the earliest commonly used cloning vectors is the pBR322 plasmid. Other cloning vectors include the pUC series of plasmids, and a large number of different cloning plasmid vectors are available. Many plasmids have high copy number, for example pUC19 which has a copy number of 500-700 copies per cell,[13] and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells.[14] Some plasmids contain an M13 bacteriophage origin of replication and may be used to generate single-stranded DNA. These are called phagemid, and examples are the pBluescript series of cloning vectors. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia. Link- http://en.wikipedia.org/wiki/Main_Page
Views: 98015 Shomu's Biology
PCR Cloning
 
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( http://www.abnova.com ) - PCR cloning is a method of cloning which dramatically reduces the time and effort put into the cloning reaction. PCR cloning procedure consisting of the four following steps: (1) production of a fragment of the gene using PCR, (2) digestion of genomic DNA, (3) ligation into a plasmid vector, and (4) transformation into bacteria and then bacteria will replicate the plasmid. More videos at Abnova http://www.abnova.com
Views: 46307 Abnova
Simply Cloning - Chapter 4 - Gel Purification
 
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Simply Cloning is a video manual for making DNA constructs. Chapter 4 describes how to separate DNA fragments on agarose gel, and purify them with a gel extraction kit. Narration Script: During the gel purification we are going to load the PCR fragment and the digested vector on an agarose gel, apply electric current, and separate DNA molecules based on their molecular weight. Then we will cut out DNA bands from the agarose and clean them with a gel purification kit. Gel purification allows us to separate vector DNA from uncut fraction, clean PCR fragment from unspecific PCR products and primers, and also serves as a quality control for the molecular weight of vector and insert. The summary of gel purification protocol is presented here. Please refer to the Protocols poster for additional details. Gel purification protocol: Prepare 1% agarose gel Add 1 µg of agarose to 100 ml of TEA buffer Boil in a microwave until the agarose completely dissolved Add 2 µl ethidium bromide or GelRedTM Pour into a casting tray Leave for 30 min to solidify Load the gel Molecular weight marker 1 µl of undigested vector Vector restriction digest Insert restriction digest Run the gel until bromophenol blue dye reaches the end Cut out desired DNA fragments Purify with a gel purification kit The advantage of doing vector restriction digest in parallel with PCR is that they both are ready about the same time for gel purification. This way we can run both vector and insert on the same agarose gel. Let's prepare a 1% agarose gel. I am going to weigh 1.5 grams of agarose and add it to 150 ml of TAE buffer. Then I heat it up in a microwave until it boils. A safety note: please wear heat-protective gloves when you handle a bottle with boiling agarose. Leave the screw cap on the bottle somewhat loose to allow for vapours to vent. Then I let the agarose to cool down for 15 min. Add ethidium bromide. Swirl the bottle to mix up ethidium bromide. Another safety note: remember that ethidium bromide is a cancerogen and change your gloves each time when you get in contact with gels, trays or any other contaminated equipment. At the moment many laboratories are switching to Gel Red TM (Biotium Inc) - a DNA dye that produces superb DNA staining without being cytotoxic or mutagenic. Now I am going to pour the agarose in a tray with a comb and let it solidify for 30 minutes. Finally, I am going to transfer the gel into a running tray and filled with the TAE buffer. By now both vector restriction digest and the PCR are ready. So, on the agarose gel I am going to run: Molecular weight marker 1 µl of uncut vector entire vector restriction digest and all 50 µl of PCR reaction To each of these three tubes (except the marker) I have already added 5 ul of the loading dye, which contains bromophenol blue and glycerol. I am back to the gel room with my tubes and I am ready to load the gel. Once again: molecular weight marker... uncut plasmid... cut plasmid... and the PCR product. I will run the gel for 35 min at 105 V. Remember, that DNA is negatively charged and therefore it will run towards the positive, or red electrode. The run is over, let's take the gel to a UV transilluminator and have a look at it. Here is our DNA separated on the agarose gel. Let me put some labels here. In the left lane we have the marker, then uncut vector, cut vector and the PCR product. There are few pieces of information that I extract from this kind of picture. First of all, I compare uncut and cut vector lanes. You see how the entire vector band shifted. This means that the vector was linearized. I also look at the molecular weight of the linearized vector and compare it with the information from the sequence file. The size ofpSAT6-MCS is supposed to be 3900 base pairs, which is in agreement with what I see on the gel. In addition I roughly estimate the amount of vector DNA. The rule of thumb is that if you can see it well on the gel, then you should have sufficient amount for efficient ligation. In the case of the PCR product I also look at the molecular weight and at the amount of DNA. Right now everything looks great, so I will proceed with cutting out the vector and the PCR fragment from the gel. Before cutting the gel I prepare two labelled Eppendorf tubes, two razor blades, and a UV shield. Now I am going to put the UV shield on, pull out the transilluminator from the imaging system and turn on UV light.
Views: 20723 Andriy Nemirov
Agarose Gel Electrophoresis - Amrita University
 
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▶ This video channel is developed by Amrita University's CREATE http://www.amrita.edu/create ▶ Subscribe @ https://www.youtube.com/user/amritacreate http://www.youtube.com/amritavlab ▶ Like us @ https://www.facebook.com/CREATEatAmrita ▶ For more Information @ http://vlab.amrita.edu/index.php?sub=3&brch=77&sim=1375&cnt=1 ▶ Amrita Virtual Lab Project website http://vlab.amrita.edu Agarose gel electrophoresis is the easiest and commonest way of separating and analyzing DNA. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving. The purpose of the gel might be to look at the DNA, to quantify it or to isolate a particular band. The DNA is visualized in the gel by addition of ethidium bromide. This binds strongly to DNA by intercalating between the bases and is fluorescent, meaning that it absorbs invisible UV light and transmits the energy as visible orange light.
Views: 224467 Amrita Vlab
Causes of the smeared DNA bands after Gel electrophoresis and recommendations to avoid it
 
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Join Easy Experiment Academy for more lectures https://www.udemy.com/dna-primer-design-for-pcr/ Facebook page https://m.facebook.com/easyexperimentacademy/ -------------------------------------------------------- Gel electrophoresis, DNA Gel electrophoresis - Simple Animated Tutorial, Agarose Gel Electrophoresis, DNA gel electrophoresis lab demo, 2 D gel electrophoresis, Biotechnology - Gel Electrophoresis, Agarose Gel Electrophoresis of DNA fragments amplified using PCR, Principles of Gel Electrophoresis, PCR and Gel Electrophoresis , Performing Agarose Gel Electrophoresis, Gel Electrophoresis process, Gel Electrophoresis problems, Gel Electrophoresis procedure, Gel Electrophoresis principle, Gel Electrophoresis animation, Gel Electrophoresis explanation, Gel Electrophoresis steps, Gel Electrophoresis experiment, Gel Electrophoresis of dna, Gel Electrophoresis demonstration,
Views: 870 Shahed and Science
pBR322 Gene Cloning Vector Explained
 
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Why the name pBR322? What are the elements in pBR322? Function of ori, restriction enzyme and selectable markers explained. Why pBR322 is known as 'ideal vector'? http://www.biologyexams4u.com/2013/01/gene-cloning-vector.html
Views: 86468 biologyexams4u
Designing PCR and Sanger Sequencing Primers– Seq It Out #5
 
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Download the free Sanger sequencing handbook at http://www.thermofisher.com/sangerhandbook Submit your question at http://www.thermofisher.com/ask Primer design is like art. There is more than one design to cover the region of interest. Are you an “Artist”? Primers are crucial to the success of target amplification and subsequent sequencing in PCR and Sanger sequencing workflows. Let’s take a look at our lab book In the typical Sanger sequencing workflow from genomic DNA, one needs to first amplify the target by PCR, and then subsequently run the Sanger sequencing reaction. If you start from purified plasmid DNA, one only needs to run the Sanger sequencing reaction. PCR amplification requires 2 primers from opposite strands that determine the region of sequence amplified in the forward and reverse direction. Sanger sequencing differs from PCR in that only a single primer is used in the reaction. Typically, for a given PCR fragment, two Sanger sequencing reactions are set up, one for sequencing the forward strand, the other one for sequencing the reverse strand. Primer design is an important aspect relating to many forms of PCR including basic PCR, fragment analysis, quantitative analysis and Sanger sequencing.. Here are a few things to keep in mind when designing your own primers. 1. Primer length should be in the range of 18 to 22 bases. 2. The primer should have GC content of 50% to 55%. 3. Primers should have a GC-lock on the 3’ end. 4. The melting temperature of any good primer should be in the range of 50OC to 55OC. 5. The primer should not include poly base regions. 6. Four or more bases that compliment either direction of the primer should be avoided. In addition, there are some PCR specific guidelines to help you design good PCR primers. These guidelines can be found on our website. Since primer design is not easy. There is risk of designing the wrong primers which could be costly in your experiments. You may be asking: Is there an easier way? The answer is Yes, and Thermo Fisher Scientific has a free tool to help you out. (www.lifetechnologies.com/primerdesigner) Primer Designer tool is a free PCR/Sanger primer online search tool that includes over 600,000 primer pairs covering the human exome and human mitochondrial genome. You can choose the range of amplicon length for your sample and your research interest to optimize it for your experiment. If you are doing NGS confirmation with your capillary electrophoresis genetic analyzer, this tool can really simplify your workflow. You can use this online by uploading your .vcf file, or if you have an Ion Torrent sequencer, even better, as there is a seamless integration with your ion reporter software to upload your data into this Primer Designer tool. Now you can find the right primers with just a few simple clicks. I hope this video was helpful on primer design, and I am sure you’ll have more questions. Submit your question at http://www.thermofisher.com/ask and subscribe to our channel to see more videos like this. And remember, when in doubt, just Seq It Out
Primer Design and Fragment Assembly Using NEBuilder HiFi DNA Assembly or Gibson Assembly
 
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Watch an interactive tutorial on primer design to see how simple it really is to clone with either NEBuilder® HiFi DNA Assembly or the Gibson Assembly® Cloning Kit.
Views: 22551 New England Biolabs
Plasmid Mini Prep Protocol
 
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See more video protocols, please visit http://www.video-protocols.com MO BIO's UltraClean 6 Minute Mini and Standard Mini Plasmid Prep Kit - Detailed Protocol, including a description of "What's Happening" at critical steps.
Views: 10127 VideoProtocols Com
Troubleshooting a Bad PCR
 
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Synthetic Biology One is a free, open online course in synthetic biology beginning at the undergraduate level. We welcome scientists, artists, journalists, policymakers, or anyone interested in designing with DNA. Meet us at syntheticbiology1.com!
Restriction enzymes
 
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Visit us (http://www.khanacademy.org/science/healthcare-and-medicine) for health and medicine content or (http://www.khanacademy.org/test-prep/mcat) for MCAT related content. These videos do not provide medical advice and are for informational purposes only. The videos are not intended to be a substitute for professional medical advice, diagnosis or treatment. Always seek the advice of a qualified health provider with any questions you may have regarding a medical condition. Never disregard professional medical advice or delay in seeking it because of something you have read or seen in any Khan Academy video.
Views: 244003 khanacademymedicine
Topo cloning
 
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Learn about topo cloning with this video. http://shomusbiology.weebly.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html Source of all articles published in description is Wikipedia. Thanks to original content developers. Link- http://en.wikipedia.org/wiki/Main_Page TOPO cloning is a molecular biology technique in which DNA fragments amplified by Taq polymerase are cloned into specific vectors without the requirement for DNA ligases. The Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosin (A) to the 3'-end of the PCR products. This characteristic is exploited in TOPO-cloning. The technique utilises the inherent biological activity of DNA topoisomerase I. The biological role of topoisomerase is to cleave and rejoin supercoiled DNA ends to facilitate replication. Vaccinia virus topoisomerase I specifically recognises DNA sequence 5´-(C/T)CCTT-3'. During replication, the enzyme digests DNA specifically at this sequence, unwinds the DNA and re-ligates it again at the 3' phosphate group of the thymidine base. TOPO vectors are designed in such a way that they carry this specific sequence 5´-(C/T)CCTT-3' at the two linear ends. The linear vector DNA already has the topoisomerase enzyme covalently attached to both of its strands' free 3' ends. This is then mixed with PCR products. When the free 5' ends of the PCR product strands attack the topoisomerase/3' end of each vector strand, the strands are covalently linked by the already bound topoisomerase. This reaction proceeds efficiently when this solution is incubated at room temperature with required salt.[1] Different types of vectors are used for cloning fragments amplified by either Taq or Pfu polymerase as Taq polymerase (unlike Pfu) leaves an extra "A" nucleotide at the 3'end during amplification. The TA TOPO cloning technique relies on the ability of adenine (A) and thymine (T) (complementary basepairs) on different DNA fragments to hybridize and, in the presence of ligase or topoisomerase, become ligated together. The insert is created by PCR using Taq DNA polymerase, a polymerase that lacks 3' to 5' proofreading activity and with a high probability adds a single, 3'-adenine overhang to each end of the PCR product. It is best if the PCR primers have guanines at the 5' end as this maximizes probability of Taq DNA polymerase adding the terminal adenosine overhang. Thermostable polymerases containing extensive 3´ to 5´ exonuclease activity should not be used as they do not leave the 3´ adenine-overhangs. The target vector is linearized and cut with a blunt-end restriction enzyme. This vector is then tailed with dideoxythymidine triphosphate (ddTTP) using terminal transferase. It is important to use ddTTP to ensure the addition of only one T residue. This tailing leaves the vector with a single 3'-overhanging thymine residue on each blunt end.
Views: 19021 Shomu's Biology
Gel Graphing.mp4
 
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A simple explanation on how to generate a graph of an agarose gel
Views: 28369 Kirk Brown
PCR based cloning
 
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Perform PCR based cloning using Serial Cloner
Views: 8911 Siddharth Srivastava
Simply Cloning - Chapter 1 - Planning
 
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Simply Cloning is a video manual for making DNA constructs. Chapter 1 deals with experiment planning, building plasmid maps in Vector NTI, and primer design Narration Script: In this section I am going to demonstrate how to create experiment outline in PowerPoint, build plasmid maps, and design primers for your cloning project. I begin each of my cloning projects by making a PowerPoint file with a project outline. For that I open a new PowerPoint file and save it as pSAT6-Bar, which is the project name. I change the layout to my favourite, which is "Title Only". And I put the project name on top. On this slide I put the name of my starting plasmid, which is pSAT6-MCS. Let's add some style to it. Then I draw a down arrow, add some style to it as well, and put the name of the plasmid I am trying to make, which is going to be pSAT6-Bar. For this one I will use a new style, which is going to be black. Also on this slide I want to have a name of my PCR template, which is a plasmid called pFGC 5941. Let's give it some style as well. Also I want to have the names of the primers I will be using, which is Bar-XhoI-F and Bar-BamHI-R. I will draw another arrow indicating that PCR product will be inserted into pSAT6-MCS. And, finally, I will indicate the restriction enzymes I will be using to cut my Vector and Insert. A usual cloning project goes for several days and in many cases you will be running several of those projects at a time. That's why I would recommend printing out this page and keeping it somewhere nearby, so when you come to your lab in the morning you know exactly where you left off. Let's move to the next part of the planning stage, which is building vector maps and designing primers. The software package I will use in this chapter is Vector NTI from Invitrogen. In Supplement 3, which is at the end of this video, I will demonstrate how to do exactly the same vector map design with a free software pDRAW32. I start with two browser windows, one displaying the sequence of pSAT6-MCS, another - sequence of pFGC5941. I launch Vector NTI Explorer, which keeps track of all the maps I have in the database. I create a new database subset, label it as Bar, and click on this icon, which says New. I name the molecule as pSAT6-MCS, indicate that it is circular, and open Edit Sequence window. Then I go back to the web browser, select and copy the sequence of pSAT6-MCS, and paste it in the Edit Sequence window. Let's look at this sequence in Vector NTI. Vector NTI opens with three panes. On the top right you see the map, on the bottom is the sequence, and on the top left is the information about the molecule. If you click on the Restriction/Methylation Folder you will see a list of all restriction enzymes currently displayed on the map. Now, let's add the features of pSAT6-MCS to this map. For that I click on ADD FEATURE, switch to the web browser window and select Feature View. You can see here a list of all pSAT6 features with their coordinates. Let's transfer them to our map. In the ADD FEATURE menu I am going to enter feature name, which is 2x35S Promoter, and starting and ending point. In the Vector NTI I could also pick different feature types, which reflect how the features are displayed on the map. What I particularly like about VNTI is that as I am adding those features, I can see them both on the map and on the sequence. Now I will proceed adding the rest of the features, namely Multiple Cloning Site, Terminator and Ampicillin resistance gene. Again, I get all the information about feature locations from the PubMed Nucleotide file. pSAT6-MCS map is ready, I am going to save it and move onto building pSAT6-Bar. I will build pSAT6-Bar by pasting the sequence of the bar gene from PubMed Nucleotide. First, I locate XhoI and BamHI sites on pSAT6-MCS and remove sequence between them. Then I will go to the PubMed Nucleotide and locate the sequence of the bar gene. When I scroll down and look in the annotations I can see that the bar gene is located on the reverse strand in the positions 373 - 924. To make life a little simpler, I will use PubMed tool called Change Region Shown. I enter here the boundaries of the bar gene and click on Update View. Now if I scroll all the way down you can see that the sequence of the pFGC5941 reduced to a 500 base pair region, which is the bar gene. Also, if you have a closer look at it, there are an ATG and a Stop codons on the complimentary strand of this sequence. So, let's copy the bar gene, go back to Vector NTI and paste this sequence between XhoI and BamHI sites in pSAT6-MCS.
Views: 51405 Andriy Nemirov
A Simple Method to find PCR Product length from Primer Sequence
 
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http://technologyinscience.blogspot.com/2013/05/a-simple-method-to-find-pcr-product.html A simple method for Calculating the PCR product length / amplicon size from the primer sequence.
Views: 22410 Bio-Resource
Simply Cloning - Supplement 1 - Troubleshooting
 
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In the case you don't get your clones from the first time, I want to suggest a couple of troubleshooting ideas. The first one deals with the case when you have no colonies on your vector plus insert plate on the next day after the transformation. In most cases the lack of colonies on the vector plus insert plate can be tracked to lost DNA, either of the vector or of the insert. To test that, I prepare an agarose gel and run 5 µl each of the vector and of the insert DNA. On this gel picture you can see relatively intense bands for both vector and insert. However, if one of your DNA fragments is barely visible or missing, it would most likely cause low efficiency of ligation. If that's the case, try to identify a step where you lose the DNA, or prepare the DNA fragment again. Another reason for not having any colonies on Day 2 is the low quality of plasmid DNA. An easy way to check the quality of plasmid DNA is to retransform 1 µl of the plasmid into competent cells. If you get a carpet of colonies like the one on this plate, then you plasmid DNA is good. However, if you get just 10 - 20 or no colonies, it would indicate the low quality of plasmid DNA. In such a case, pick one of those colonies, prepare fresh plasmid miniprep, and try to clone your insert into the fresh plasmid.
Views: 3011 Andriy Nemirov
PCR - Polymerase Chain Reaction (IQOG-CSIC)
 
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PCR technique (Polymerase Chain Reaction), Animation. It is a technique used to make multiple copies of a segment DNA of interest, generating a large amount of copies from a small initial simple. Amplification of DNA segments makes possible the detection of pathogenic virus or bacteria, identification of individuals (DNA fingerprinting), and several scientific research involving DNA manipulation. Spanish version: http://youtu.be/TalHTjA5gKU This video has been produced in the Institute of General Organic Chemistry of the CSIC (IQOG-CSIC), Spain, by Guillermo Corrales, as part of its task for promoting Science Communication and may be freely used for educational and science popularization purposes. Canal Divulgación. Divulgación científica. Instituto de Química Orgánica General (QOG) CSIC Madrid, Spain http://www.youtube.com/user/CanalDivulgacion
Views: 1788144 CanalDivulgación
Key Steps of Molecular Cloning
 
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Sound track link http://incompetech.com/music/royalty-free/?keywords=tech+talk Script: Molecular cloning is a process of isolation of a specific DNA fragment and transfer of this fragment into a plasmid vector. As a part of the plasmid vector, the DNA fragment could be easily amplified, sequenced, stored for long periods of time, and used for gene expression and other functional studies. Two starting ingredients of molecular cloning are a plasmid vector and a DNA fragment that has to be inserted in it. The DNA fragment is usually a gene or other functional region from a living cell, or it could be an artificial sequence with properties useful for a researcher. Plasmid vector is circular piece of DNA that could be easily amplified in E. coli, stored for long periods of time, and easily manipulated in a test tube. A typical plasmid vector contains: - an origin of replication that allows it to be replicated inside a bacterial cell; - a selection marker, for example a beta lactamase gene coding for ampicillin resistance; - and a multiple cloning site , which could be cleaved with several restriction enzymes, such as BamHI (G-GATCC), EcoRI (G-AATTC) or NcoI (C-CATGG). In many vectors, the multiple cloning site is surrounded by sequences of promoter and terminator, that guide expression of inserted genes after the vector is introduced inside a cell. To be used for molecular cloning, both vector and insert DNA are treated with restriction enzymes that cleave double stranded DNA molecules producing overhanging single stranded nucleotide tails. After their ends have been prepared with restriction enzymes, vector and insert are combined together, and another enzyme, called a DNA ligase is added to the mix. At the same time as complimentary base pairing of single stranded overhands brings the ends of vector and insert together, the DNA ligase fuses them into one intact DNA molecule. In order to make multiple copies of this molecule, the ligation mixture is introduced inside the E. coli cells in a process called transformation. During the transformation the cell-DNA mixture is kept on ice and then exposed to 42 oC. Such sudden change in temperature drives the DNA inside some of the E. coli cells. Then the cells are plated on a plate with growth medium supplemented with a selective antibiotic. Only the cells that acquired the plasmid have resistance to the antibiotic and are capable of growth on such a medium. After overnight incubation at 37 oC each transformed cell produces a colony of identical cells, oftentimes called a clone. The selected clones are then individually picked, grown even further in a liquid medium, and the DNA is extracted from them. Thus, in the process of molecular cloning, a DNA fragment that represented a tiny fraction of cell genome is integrated into a bacterial plasmid. As a part of a plasmid, this DNA fragment represents a quarter or more of total DNA in a test tube and it could be effortlessly and endlessly amplified in E. coli.
Views: 238718 Andriy Nemirov
Competent Cell Transformation
 
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Visit http://www.invitrogen.com/compcells for more information. Overview of chemical transformation This video will walk you through the basics of chemical transformation. Transformation is the process by which bacterial cells take up foreign DNA from their environment. Typically, this is done in the lab for two main reasons: to propagate a recombinant plasmid or to obtain the results of a sub-cloning reaction. There are two main classes of competent cells: chemical and electro-competent. The procedure shown in this video can be used with most chemically competent cells. We are focusing on the protocol provided with Invitrogen's Top 10 strain of comp cell. Getting Started First, let's talk about storage conditions and what comes with your kit. Competent cells are stored at minus 80° degrees. Invitrogen's kits come with vials of competent cells, transformation instructions, a vial of S.O.C. medium, and a pUC19 transformation control. The OneShot® Top 10 kit used in this video comes with vials containing 50 micro liters of competent cells; enough for one transformation per tube. The competent cells must remain frozen until just before you are ready to use them. If they thaw too soon, it can affect the transformation efficiency. For your transformation protocol, you will need the following items: - A water bath set at 42° degrees - An ice bucket with ice - A 37° degree shaking incubator - A 37° degree incubator - 10 cm diameter LB Agar plates with the appropriate antibiotic In this case a 100 micrograms per microliter of ampicillin The selective plates should be warmed in an incubator for 30 minutes before you use them. You will need one plate for each transformation. Finally, you will need the vial of S.O.C. medium thawed and at room temperature. The first step is to mix the comp cells and the plasmid DNA of interest. Briefly centrifuge the DNA and put it on ice. This DNA can be from a ligation reaction or plasmid DNA you wish to propagate. Next, thaw on ice one 50 microliter OneShot™ vial of cells for each transformation reaction. Pipette one to five microliters of your DNA samples directly into each vial of competent cells. Mix by tapping gently and not by pipetting up and down. Your remaining ligation reaction can be stored at minus 20° degrees. We also highly recommend setting up a transformation control with the PUC 19 provided to ensure the transformation was performed correctly and the competent cells were at the expected competency. Add one microliter of the pUC19 control plasmid to 50 microliters of competent cells and tap gently to mix. The next step is to incubate the cells on ice for 30 minutes. Next is the heat shock step. This is harsh on the cells, so be sure not to mix or shake the vials. Incubate for exactly 30 seconds in the 42° degree water bath. After the incubation, remove and place on ice. Now the cells are allowed to recover from the transformation and are grown in rich S.O.C. medium. Add 250 microliters of the pre-warmed S.O.C. medium to each vial. Next, place the vials in a microcentrifuge rack inside the shaking incubator. Or, tape the vials on their sides to the shaking platform in the incubator. Shake the vials at 37°C for one hour at 225 rpm. If you do not have a shaking incubator you can add the vials to a 37°C regular incubator. Once the cells have recovered, it is time to plate them. The antibiotic you added will select only transformed cells. Pipette 15 microliters from the individual vial onto its own labeled LB plate. The remaining transformation mix may be stored at 4° and plated the next day if desired. For the control, remove 10 microliters from the vial and mix with 20 microliters of fresh S.O.C. and plate the entire 30 microliters as you did with the other transformations. To spread the cells evenly across the plate add 5 to 8 sterile glass beads and rotate, or use a sterile glass rod to spread across the plate. After removing the glass beads, invert the plates and incubate in 37° degrees overnight. Plasmid containing the antibiotic resistance marker that allowed them to grow in the presence of a selection antibiotic; the pUC19 control plate will have a larger number of colonies than your ligation plates. Many people also use blue white screening for selecting their clones. Each of these is a transformant and has taken up the plasmid containing the antibiotic resistance marker that allowed them to grow in the presence of the selection antibiotic. The pUC19 control plate will have a larger number of colonies than your ligation plates. Many people also use blue white screening for selecting their clones. For more trouble shooting tips or information on all competent cell structures visit http://www.invitrogen.com/compcells
Genetics: how to read Agarose gel electrophoresis running results
 
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Attention - just noticed after uploaded video that picture is not very good and some think that band C is on top of the letter C some - that it is to the right and some that it is below (I personally sought it is on top) so it cause a lot of confusion - but at least theory should help you understand how gel electrophoresis works) Gel electrophoresis is the standard lab procedure for separating DNA by size (e.g. length in base pairs) for visualization and purification. Electrophoresis uses an electrical field to move the negatively charged DNA toward a positive electrode through an agarose gel matrix. The gel matrix allows shorter DNA fragments to migrate more quickly than larger ones. Thus, you can accurately determine the length of a DNA segment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of known lengths). What is a Gel? You may be wondering what exactly a gel is, and what it has to do with agarose. Let's find out by "making" a gel. Purified agarose is in powdered form, and is insoluble in water (or buffer) at room temperature. But it dissolves in boiling water. When it starts to cool, it undergoes what is known as polymerization. Rather than staying dissolved in the water or coming out of solution, the sugar polymers crosslink with each other, causing the solution to "gel" into a semi-solid matrix much like "Jello" only more firm. The more agarose is dissolved in the boiling water, the firmer the gel will be. While the solution is still hot, we pour it into a mold called a "casting tray" so it will assume the shape we want as it polymerizes (otherwise it will just solidify in the bottom of the flask wasting the expensive agarose). Look through the sequence of images below to learn how to prepare a gel. [AGAROSE ON SCALE] [BUFFER] [microwave] [CASTING TRAY] [POURING] [polymerized gel] [buffer in tank] How are Gels Loaded and Run? Imagine you are a DNA molecule. If you were inside an agarose gel, your environment would resemble a very dense spider web. If you are a small fragment, you could easily crawl through the spaces in between the webs (they are too tough for you to just pull out of the way). But as you increase in length, it gets harder and harder for you to fit through the spaces. If it were a race between you and another DNA molecule, who would win? Do you think the same would hold true for any charged molecule? Now it's time to take the DNA we digested in Experiment 1 and load it on the gel we just prepared. So again, follow through the pictures below to load and run our gel. Congratulations! You have now digested a piece of DNA with Restriction Enzymes, separated the digested fragments by Agarose Gel Electrophoresis on a gel you poured, and analyzed and documented your results. And you don't even have to clean up after yourself....
Primer Design for PCR
 
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In this lecture, I explain how to design working primers for use in PCR. If you are unfamiliar with PCR, watch the following video: http://www.youtube.com/watch?v=2KoLnIwoZKU. Created by:Tyler Maxfield
Views: 324710 Herbert Sauro
PCR Basics
 
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00:00 Introduction to polymerase chain reaction (PCR) 01:04 Primers 02:34 Primers interact with templates 03:07 Three steps of PCR 03:55 Denature 04:55 Anneal 06:15 Extension; DNA polymerase 07:57 Positions of primers define the length of the amplicon 09:42 "Big picture" view of PCR cycling over time 14:18 Uses of PCR in the study of genetics 14:57 Details of a setting up a PCR experiment 20:27 PCR is sensitive to contamination
Views: 34642 Joseph Ross
Gateway® Cloning Technology
 
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Visit http://www.lifetechnologies.com/gateway Gateway® recombination cloning technology circumvents traditional restriction enzyme based cloning limitations, enabling you to access virtually any expression system in just a few simple steps. From cloning or sub-cloning of DNA fragments over protein expression to functional analysis, Gateway® cloning technology is applicable for a variety of research areas, for truly multidisciplinary scientific studies and get's you there faster than with traditional methods. ------------------ Audio transcript: The typical cloning workflow involves many steps, takes a considerable amount of time and even then success is not guaranteed. Imagine a cloning workflow which was fast, flexible, efficient, and delivered consistent results. Well welcome to Gateway Cloning Technology. Regardless of the desired end result—cloning or subcloning of DNA fragments, protein expression, or gene analysis— Gateway Cloning Technology gets you there faster than traditional methods. Here's how it works. First, set up the reaction by adding the starting DNA material, buffer and clonase enzyme to your microfuge tube. Next, incubate at room temperature for one hour. Then add competent cells to your mixture and allow 45 minutes for the transformation to take place. After transformation, plate the reaction and incubate overnight at 37 degrees celcius. It is that simple! Gateway Cloning Technology gives you: • Fast, one hour, room temperature cloning reactions with greater than 99% efficiency. • Maintains orientation and reading frame without using restriction enzymes or ligation. • Eliminates re-sequencing ensuring consistent results throughout your experiment using the same clone from target identification to validation. • Shuttles DNA from one entry clone to multiple expression vectors, affording flexibility while simplifying your cloning workflow. Discover the versatility of Gateway Cloning Technology. Visit lifetechnology.com/gateway today.
ApE PCR primer analysis, product size and mutation coverage
 
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Bio125, Molecular biology and genomics, Spelman College, spring 2015 Analyze PCR primers and verify their coverage of mutation on plasmid.
Views: 1196 Hong Qin
Expression vector
 
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This lecture explains about the expression vector properties and the uses of expression vector in gene cloning. http://shomusbiology.weebly.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for protein expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The plasmid is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector.[1] The goal of a well-designed expression vector is the production of significant amount of stable messenger RNA, and therefore proteins. Expression vectors are basic tools for biotechnology and the production of proteins, for example insulin which is important for medical treatments of diabetes. An expression vector has features that any vector may have, such as an origin of replication, a selectable marker, and a suitable site for the insertion of a gene such as the multiple cloning site. The cloned gene may be transferred from a specialized cloning vectors to an expression vector, although it is possible to clone directly into an expression vector. The cloning process is normally performed in Escherichia coli, and vectors used for protein expression in organisms other than E.coli may have, in addition to a suitable origin of replication for its propagation in E. coli, elements that allow them to be maintained in another organism, and these vectors are called shuttle vector. Elements for expression Further information: Transcription (genetics) and Translation (biology) An expression vector must have elements necessary for protein expression. These may include a strong promoter, the correct translation initiation sequence such as a ribosomal binding site and start codon, a strong termination codon, and a transcription termination sequence. There are differences in the machinery for protein synthesis between prokaryotes and eukaryotes, therefore the expression vectors must have the elements for expression that is appropriate for the chosen host. For example, prokaryotes expression vectors would have a Shine-Dalgarno sequence at its translation initiation site for the binding of ribosomes, while eukaryotes expression vectors would contain the Kozak consensus sequence. The promoter initiates the transcription and is therefore the point of control for the expression of the cloned gene. The promoters used in expression vector are normally inducible, meaning that protein synthesis is only initiated when required by the introduction of an inducer such as IPTG. Protein expression however may also be constitutive (i.e. protein is constantly expressed) in some expression vectors. Low level of constitutive protein synthesis may occur even in expression vectors with tightly controlled promoters. Protein tags Main article: Protein tag After the expression of the gene product, it is usually necessary to purify the expressed protein. However, separating the protein of interest from the great majority of proteins of the host cell can be a protracted process. To make this purification process easier, a purification tag may be added to the cloned gene. This tag could be histidine (His) tag, other marker peptides, or a fusion partners such as glutathione S-transferase or maltose-binding protein. Some of these fusion partners may also help to increase the solubility of some expressed proteins. Other fusion proteins such as green fluorescent protein may act as a reporter gene. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia. Link- http://en.wikipedia.org/wiki/Main_Page
Views: 88493 Shomu's Biology
Overview of Traditional Cloning
 
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Traditional Cloning refers to the generation of DNA fragments using restriction enzymes, and their subsequent assembly and transformation. Learn more at https://www.neb.com/applications/cloning-and-synthetic-biology
Views: 9415 New England Biolabs
Designing & Ordering Primers with Vector NTI® Software
 
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Learn more at http://www.lifetechnologies.com/vectornti Design and order primers in literally 60 seconds using Vector NTI Advance® Software. Select the region to amplify. Access the primer design menu and select "amplify selection." Add 5′ and 3′ restriction sites if required to the sense and antisense primer. Add additional bases if required to the sense and antisense primer. Inspect the left pane for thermodynamic parameters such as Tm, length, and GC content. Select the product, and click "order." Add a researcher name, any desired 5′ or 3′ modifications, or change the purity. Click "add to cart" and begin the checkout process.
Restriction Cloning
 
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SnapGene tutorial showing how to simulate standard cloning procedures.
Views: 40697 SnapGene
Simply Cloning - Chapter 6 - Ligation
 
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Simply Cloning is a video manual for making DNA constructs. Chapter 6 describes how to ligate DNA fragments together using a T4 DNA ligase. Narration Script: During the ligation step we are going to mix the linerized vector and the insert together and add a T4 DNA ligase. The ligase will fuse their ends forming a circular plasmid. Ligation protocol: 1. Ligation mix o 9 µl ddH2O o 2 µl 10X ligation buffer o 2 µl of plasmid digested with restriction enzymes A and B o 6 µl of insert digested with restriction enzymes A and B. For the ligation control, use 6 µl of water instead of insert o 1 µl DNA ligase 2. Split ligation mix into two tubes 10 µl each 3. Incubate tube 1 at room temperature for 15 min 4. Incubate tube 2 at 16 oC for 24 hours Let's set up the ligation reaction. Here on the bench I have: • Double distilled water • A tube with cut vector • A tube with cut insert • An aliquot of ligase buffer - remember that the ligase buffer has to be aliquoted to avoid freeze-thawing • An empty tube for vector plus insert • An empty tube for control • And the T4 DNA Ligase I am going to set up two reactions, one with vector and insert and one with vector alone for control. In the Vector plus Insert tube I am going to combine: • 9 µl of water • 2 ul of cut vector • 6 ul of cut insert • 2 µl of ligase buffer • And 1 µl of DNA ligase And as always, I mix it up and down with a pipette. The control tube is pretty much identical, the only difference is that it contains water instead of insert. In the control tube I am going to put: • 15 µl of water, it's 9 + 6 instead of insert • 2 ul of cut vector • 2 µl of ligase buffer (we are skipping the insert) • 1 µl of DNA ligase And the mixing step. Now I am going to split the contents of ligation tubes into two aliquots. Here I have two new tubes, one of them I will label as Vector Plus Insert, another one as Vector Control. And I will transfer 10 ul from each of the ligation tubes into its replica. I am going to leave these two tube on the bench for 15 minute room temperature ligation, and I will put the other two tubes in a 16 oC incubator overnight. If tomorrow I will not get any colonies from the 15 minute ligation, I will repeat the transformation step with the tubes I kept at 16 oC.
Views: 14256 Andriy Nemirov
Cloning vector
 
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This DNA and gene cloning lecture explains the properties of cloning vectors including the cloning vector uses in molecular cloning in animal and plants. http://shomusbiology.weebly.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.[1] The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use. There are many types of cloning vectors, but the most commonly used ones are genetically engineered plasmids. Cloning is generally first performed using Escherichia coli, and cloning vectors in E. coli include plasmids, bacteriophages (such as phage λ), cosmids, and bacterial artificial chromosomes (BACs). Some DNA however cannot be stably maintained in E. coli, for example very large DNA fragment, and other organisms such as yeast may be used. Cloning vectors in yeast include yeast artificial chromosomes (YACs). Plasmid is an autonomously replicating circular extra-chromosomal DNA. They are the standard cloning vectors and the most commonly used. Most general plasmids may be used to clone DNA insert of up to 15 kb in size. One of the earliest commonly used cloning vectors is the pBR322 plasmid. Other cloning vectors include the pUC series of plasmids, and a large number of different cloning plasmid vectors are available. Many plasmids have high copy number, for example pUC19 which has a copy number of 500-700 copies per cell,[13] and high copy number is useful as it produces greater yield of recombinant plasmid for subsequent manipulation. However low-copy-number plasmids may be preferably used in certain circumstances, for example, when the protein from the cloned gene is toxic to the cells.[14] Some plasmids contain an M13 bacteriophage origin of replication and may be used to generate single-stranded DNA. These are called phagemid, and examples are the pBluescript series of cloning vectors. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia. Link- http://en.wikipedia.org/wiki/Main_Page
Views: 85218 Shomu's Biology
Biology Biotechnology Principles part 13 (Vectors: Restriction Sites) class 12 XII
 
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Biology Biotechnology Principles part 13 (Vectors: Restriction Sites, Origin of Replication) class 12 XII
Views: 66085 ExamFear Education
Gel Extraction
 
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( http://www.abnova.com ) - Gel extraction is a technique used to isolate a desired fragment of intact DNA from an agarose gel following agarose gel electrophoresis. Three basic steps are involved: slicing the bands of interest on UV light exposure, isolating the DNA from those bands, and removing the accompanying salts and stain. More videos at Abnova http://www.abnova.com
Views: 24938 Abnova
Why Are Plasmids Essential For Recombinant DNA Technology?
 
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Restriction enzyme sites in non essential regions technology. Making recombinant dna an introduction to genetic analysis cloning with plasmid vectors molecular cell biology ncbi structural biochemistry techniques plasmids technology. Googleusercontent search. Application of recombinant dna technology to the springer linkthe official blog edvotekrecombinant an overview. Recombinant dna technology has enabled many important recombinant joining together of molecules from two the most commonly used vectors are plasmids (circular that a plasmid is small molecule within cell physically separated essential genetic information for living under normal conditions, drive replication sequences host organisms after covering basic techniques in and pcr, their do not encode an function to bacterium, which features. Plasmid vectors and viruses which are useful for recombinant dna essential low copy number plasmidscolirecombinant products human plasmids molecules of that found in bacteria separate from the basis a laboratory introduction to technology every why technology? A. Chapter 20 sapling flashcards. The cloning of specific dna fragments usually involve 1) insertion into a vector (a recombinant vector)coli and plasmid vectors are in common use because they technically technology also referred to as molecular is these vectors, foreign inserted sequence that encodes an essential part 3. Recombinant dna technology isolating and biol4900 essential features of plasmid vectors memorial universityboundless microbiology. Plasmids are small circular dna molecules found in recombinant technology v. Quizlet quizlet 112150491 chapter 20 sapling flash cards url? Q webcache. A cis acting small dna segment is essential and sufficient for plasmid partition, 18 jul 2014 posts about recombinant technology written by drsnowflack. Two enzymes are used to produce recombinant plasmids structural biochemistry dna techniques plasmid in technology, based reporter gene crucial as they allow easy manipulate and isolate using bacteria (see also alkaline lysis) can be this tiny but mighty molecule is the basis of technology. Replication of plasmid dna and its partition to daughter cells. Application of recombinant dna technology to the production useful biomaterials. Dna recombinant techniques plasmid. Dna recombinant techniques plasmid chapter 20 sapling flashcards addgene what is a plasmid? . In addition to their chromosomal dna, many bacteria possess extra, non essential genes today, scientists use these plasmids for genetic engineering gel electrophoresis is recombinant dna technology. Developing why are plasmids essential for recombinant dna technology? Dna from a gene of fragments double stranded that typically carry ge
Views: 27 Green Help
Ti and Ri Plasmid
 
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This online lecture tell you about Ti, Ri plasmid and it,s T-DNA Hope this lecture would be good for all viewers. Thank..........
Views: 827 Jaiki biology
Transformation of E. coli with Plasmid DNA - Edvotek Video Tutorial
 
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For more information on transformation, check out our Quick Guide! http://www.edvotek.com/Transformation_Guide.pdf In the laboratory, scientists can force bacteria like E. coli to acquire exogenous DNA from the surrounding environment through a process called transformation. The newly acquired genetic information is both stable and heritable. Kits featured in this video: http://www.edvotek.com/223 http://www.edvotek.com/221 © 2013 Edvotek Inc. All rights reserved. www.edvotek.com
Views: 31286 Edvotek Inc.
Ligation
 
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THIS IS A GOOD VIDEO TO WATCH BEFORE DOING PCR. It mentions a lot of good tips for working with reagents and protocols. Specifically, this video goes over the Promega ligation protocol for Assignment 2. ***Note: In the video, Dr. Udall makes a control reaction following the "control" reaction mix on the protocol table. For the class, students will make a control reaction following the "background" reaction mix instructions. The background does not contain any DNA, just the vector. The ligation mix he makes is called "standard" in the protocol. This is a misnomer - be sure to refer to this experimental ligation mix by a more appropriate name.
Views: 2873 PWS310TA
What is PRIMER WALKING? What does PRIMER WALKING mean? PRIMER WALKING meaning & explanation
 
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What is PRIMER WALKING? What does PRIMER WALKING mean? PRIMER WALKING meaning - PRIMER WALKING definition - PRIMER WALKING explanation. SUBSCRIBE to our Google Earth flights channel - http://www.youtube.com/channel/UC6UuCPh7GrXznZi0Hz2YQnQ?sub_confirmation=1 Source: Wikipedia.org article, adapted under https://creativecommons.org/licenses/by-sa/3.0/ license. Primer walking is a sequencing method of choice for sequencing DNA fragments between 1.3 and 7 kilobases. Such fragments are too long to be sequenced in a single sequence read using the chain termination method. This method works by dividing the long sequence into several consecutive short ones. The DNA of interest may be a plasmid insert, a PCR product or a fragment representing a gap when sequencing a genome. The term "primer walking" is used where the main aim is to sequence the genome. The term "chromosome walking" is used instead when the sequence is known but there is no clone of a gene. For example, the gene for a disease may be located near a specific marker such as an RFLP on the sequence. The fragment is first sequenced as if it were a shorter fragment. Sequencing is performed from each end using either universal primers or specifically designed ones. This should identify the first 1000 or so bases. In order to completely sequence the region of interest, design and synthesis of new primers (complementary to the final 20 bases of the known sequence) is necessary to obtain contiguous sequence information. The overall process is as follows: 1. A primer that matches the beginning of the DNA to sequence is used to synthesize a short DNA strand adjacent to the unknown sequence, starting with the primer (see PCR). 2. The new short DNA strand is sequenced using the chain termination method. 3. The end of the sequenced strand is used as a primer for the next part of the long DNA sequence, hence the term "walking". The method can be used to sequence entire chromosomes (hence "chromosome walking"). Primer walking was also the basis for the development of shotgun sequencing, which uses random primers instead of specifically chosen ones.
Views: 67 The Audiopedia
Polyacrylamide Gel Electrophoresis- PAGE - Amrita University
 
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▶ This video channel is developed by Amrita University's CREATE http://www.amrita.edu/create ▶ Subscribe @ https://www.youtube.com/user/amritacreate http://www.youtube.com/amritavlab ▶ Like us @ https://www.facebook.com/CREATEatAmrita ▶ For more Information @ http://vlab.amrita.edu/index.php?sub=3&brch=186&sim=319&cnt=1 ▶ Amrita Virtual Lab Project website http://vlab.amrita.edu PAGE (Polyacrylamide Gel Electrophoresis), is the most widely used analytical method to resolve separate components of a protein mixture based on size. For this protein molecules of different shapes and sizes, need to be denatured. This is done with the aid of SDS, so that the proteins no longer have any secondary, tertiary or quaternary structure. The proteins covered by SDS are negatively charged. When loaded onto a gel matrix and placed in an electric field, the proteins will migrate towards the anode (positively charged electrode). They are then separated by a molecular sieving effect based on size. After visualization by a protein-specific staining technique, the size of a protein can be estimated by comparison of its migration distance with that of a known molecular weight marker.
Views: 111596 Amrita Vlab
Finding the Right Primers: Using NCBI for RT-PCR Primer Design
 
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Specializing in the design and troubleshooting of qPCR, mutagenesis, and cloning experiments at IDT, Scientific Applications Specialist, Adam Clore PhD, speaks about the intelligent selection of PCR primers. Topics include identifying transcript variants for your target of interest and selecting primers that amplify only specific transcripts. Adam also discusses the growing SNP database and the potential impact of SNPs on qPCR data, how to locate any SNPs that fall in your target amplicon, and the use of free NCBI and IDT tools to help you avoid them. About the Speaker: Adam Clore Adam Clore is a Scientific Applications Specialist at Integrated DNA Technologies. He received his BS in Microbiology and Chemistry from the University of Northern Iowa and his PhD in Biology from Portland State University where he studied the molecular genetics of Hyperthermophilic Archaea and their viruses. His postdoctoral research involved studying DNA damage from environmental toxins and repair pathways. His roles at IDT include assay design and troubleshooting, new product development, research, and scientific writing.
Monarch PCR & DNA Cleanup Kit Protocol
 
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Learn how to isolate DNA from your enzymatic reactions using the Monarch PCR & DNA Cleanup Kit (5 µg). Find a written protocol for this kit at https://www.neb.com/protocols/2015/11/23/monarch-pcr-and-dna-cleanup-kit-protocol
Views: 406 New England Biolabs
How to transform E.coli via heat-shock method
 
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Transformation of competent E.coli cells via the heat-shock method. Reagents: LB Broth (1L): Tryptone - 10g Yeast Extract - 5g Sodium Chloride - 10g Mix, add DI H2O, autoclave, cool and use or keep in sealed autoclaved containers until needed. LB Agar (1L): Peptone -10g Yeast Extract - 5g Sodium Chloride - 10g Agar - 12g Dissolve in DI H2O, autoclave, wait until cool to the touch, add proper concentration of antibiotic if needed, pour 10-20ml per plate, allow agar to cool and harden. Use, or seal and keep at +4 until needed Protocol: 1. Thaw cells and DNA on ice (15-20min). 2. Mix DNA with cells, incubate on ice 15 min. 3. Heat shock at 42° C for 2 min. 4. Cool on ice for 3 min. 5. add 100ul LB, shake for 1 hour at 37° C. 6. Spread on plate 7. Place in incubator overnight at 37° C Any questions do not hesitate to ask. It is better to ask than waste reagents doing blind experiments
Views: 14969 bcburdoa
What Is The Function Of The Forward And Reverse Primers?
 
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The one made on the other strandthe primer that forward attaches to start codon of template dna (the sense strand), while reverse stop complementary 5 sep 2010 what's difference between and primer? I don't get it 14 may 2012 pcr amplification requires 2 primers determine region sequence amplified in direction. Au sparqpcr url? Q webcache. The forward primer is complementary with the top strand (read from left to right) and reverse lowest right left) if you wout do a pcr, need enhance both strands, so for one strand, called primer, which beginning of your gene, an other that will begin (in 5' end), it's. Again seems a basic question but i can't find straightforward answer anywhere how would you define difference between forward and primer is short strand of rna or dna (generally about 18 22 bases) that serves as diagrammatic representation the reverse primers for has to be complement given cdna best since double stranded complimentary, need nonsense in picture above, anneals template ( ) strand, identical (a part of). Forward & reverse primers molecular biology protocol online. So the forward primer would simply be 5' ata 3', that's easy. The one made on the other strandthe primer that polymerase chain reaction (pcr) labce, laboratory what's difference between forward and reverse selection guidelines good primers important for pcr what is a sequencing primer? The page genbank submissions handbook ncbi designing genamics. You can call them also sense and antisense primers are small dna sequences that designed to start replication in a laboratory technique called polymerase chain reaction, or pcr, amplify 6 nov 2013. Most polymerases function best at the temperatures that their cells operate (eg. What are forward and reverse primers? Forward reverse, sense antisense primers youtube. ' 3and the newly made strand will be made in the opposite direction as. The reverse primer, if i just take it from there 25 jan 2000 the purpose of sequencing is to determine order nucleotides a gene has be sequenced with forward and primers. The polymerase chain reaction (pcr) university of queensland how do forward and reverse primers work? Quora. And the reverse primer anneals to. Lastly, the forward and reverse primers should not be complementary, as a result, main difference between is direction in which they initiate replication. 20 nucleotides in length, ideallythe bankit submission tool primers page is where you will the sequence of the forward and reverse pcr primers for each the positions of the currently selected forward and reverse primers in the tables on the the parameters and their functions are outlined in the table below now let's say i want a three bp primer. Molecular biology manual primer design for a gene on the reverse principle of sequencing ugent. Biotechniques molecular biology techniques forums view topic primer (molecular biology) wikipedia. Googleusercontent search. Forward & reverse primers molecular biology protocol online the polymerase
Views: 243 hi bulbul