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How to Perform a PCR Purification
 
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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!
Views: 6685 Synthetic Biology One
PCR Purification
 
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( http://www.abnova.com ) - Following PCR, you need to get rid of excess short primers, dNTPs, enzymes, short-failed PCR products and salts. We use a silica-gel-membrane for binding of DNA in high-salt buffer (pH 4.5~5.5) and elution of DNA with low-salt buffer (pH 7.0~9.0) or ddH2O to get clean PCR products for downstream applications. More videos at Abnova http://www.abnova.com
Views: 14629 Abnova
Visual protocol on DNA cleanup with QIAquick
 
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Watch our cleanup video protocol with useful tips and tricks to help improve your downstream results. To see our full range of cleanup products please go to www.qiagen.com/cleanup. For further useful tips and tricks in end-point PCR check out: www.qiagen.com/pcrbeginnersguide. Please feel free to share this video with friends and colleagues.
Views: 21527 QIAGEN
Wizard® SV Gel and PCR Cleanup System - vacuum purification of PCR product
 
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http://www.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/ : Quickly and easily purify DNA from PCR product using a vacuum manifold
Views: 3752 Promega Corporation
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: 20892 Andriy Nemirov
Proper PCR Cleanup before Sanger Sequencing - Seq It Out #12
 
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Learn more about ExoSAP-IT Express at http://www.thermofisher.com/exosapit Why is a clean template for your Sanger sequencing reaction important? And what are the ways to get it without losing too much precious samples? Let’s find out today. The sequencing workflow usually starts with PCR also known as Polymerase Chain Reaction, where the target fragment is replicated into thousands of copies under a controlled condition using primers, DNA polymerases and deoxynucleotides. Before the PCR product is ready for Sanger sequencing, you must do one more thing, a PCR clean up step. Because Sanger sequencing is a highly accurate technique for you to read DNA sequence base by base, it is very important to clean up your reaction mixtures so that those unincorporated primers and dNTPs won’t interfere with your results. So how do you perform this PCR clean up step and what are your options? There are several methods for PCR cleanup such as ethanol precipitation, bead or column based purification, and Enzymatic approaches. Ethanol precipitation is most cost effective but is also the most labor intensive. Bead or column based purification can be an effective method for many applications. However, this route tends to be a bit pricier compared to other options. This method of purification also requires transferring samples in and out of the column which can often result in the loss of some of your precious samples so this may not be the best method for those with limited starting materials. An enzymatic cleanup approach has a really simple and straightforward workflow. In fact, it’s only one single pipetting step! It is also relatively affordable compared to the bead or column based purification methods. Basically, you add an enzyme mix to your finished PCR reaction and let it sit at a certain temperature. But to understand this method better, let’s take a look at our lab book. One of the enzymes, exonuclease I(one), digests excess primers. At the same time the other enzyme in the mix, alkaline phosphatase, dephosphorylates nucleotides in the reaction. Dephosphorylation means that the enzyme renders the dNTPs from your reaction useless, so that they don’t disturb the sequencing reaction. This is important because the sequencing reaction has a very specific ratio for its nucleotides– and you don’t want to change that ratio. Now the enzymes have done their work and you don’t need them anymore. You can simply heat up the reaction mix to 80 degrees and this will deactivate the enzymes. The result is a very clean amplicon that is now ready to go into your sequencing reaction. You don’t need to perform any other steps. And you didn’t lose any of your template either. You are done! But I am sure you’ll have more questions on PCR cleanup. Submit your question at thermofisher.com/ask and subscribe to our channel to see more videos like this. But remember, when in doubt, just Seq It Out.
How can I precipitate a PCR product (PCR purification) by easy method?
 
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Published in 11\4\2018 Easy method to purify PCR product and remove the salts and other reagent This method depend on isopropanol and sodium acetate
Practice 6 | Purification of a PCR product from an agarose gel
 
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Genetic engineering laboratory Group 1 Team 5 César Flores Daniel Martinez Jorge Nava
RapidTips PCR Purification in 60 Seconds (www.midsci.com)
 
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Visit www.midsci.com or call 800-227-9997 to order some today for your lab! RapidTip™ - PCR clean up in a tip in 60 seconds! Purification of PCR products for Sanger Sequencing in just one minute! No bind-wash-elute, enzymes, or magnetic beads necessary! An innovative new technology that removes undesirable impurities from PCR reactions leaving you with nothing but purified DNA for your Sanger sequencing applications. Primers and dNTPs will be gone in 60 seconds. ALL YOU NEED IS: Diffinity RapidTips - pre-packed with our proprietary material and ready to use out of the box! A standard pipettor - single or multi-channel. Microcentrifuge tubes to store purified DNA. 20-30µl PCR reaction volumes Purified PCR product for Sanger sequencing applications. The tip attracts and retains dNTPs, primers and primer dimers of less than 50bp and repels and permits the double stranded DNA targets (such as PCR amplicons) to remain in solution. FEATURES: Excellent Yield: Recovers up to 90% of high quality dsDNA ready to use in subsequent applications. One Minute, One Step: Extremely fast and efficient process to rapidly recover clean DNA. Cost Effective: No capital equipment (e.g., centrifuge, magnetic extractor, vacuum manifold), extra plasticware, or liquids are required. Less labor time required due to simple protocol. Robot Compatible: Easily integrated on automated equipment for high throughput processing and increased productivity. Same purification process, eliminates additional process validation. No Bind-Wash-Elute Steps: Eliminates use of large amounts of reagents and time-consuming, tedious protocols. Effective Purification: Removes up to 90% of primers, ssDNA, and primer dimers. Large Range of Fragment Length: Returns pure DNA fragments of 100bp to 10Kb. Use 20-30µl PCR reaction volumes Easy to Use: Single step protocol requires little to no training of lab personnel. Simple protocol requires no complex operator techniques or interactions with equipment, buffers, or reagents. Environmentally Friendly: Waste is limited to the functional tip. No extra materials, tips, columns, buffers, or reagents required to purify PCR product. Seamless Workflow: Uses standard pipettor so no changes are needed to your current lab workflows. HOW IT WORKS The functional pipette tip will fit on most conventional single or multiple, manual or automated laboratory pipettors. Samples containing DNA and impurities are aspirated into the tip where the impurities are adsorbed onto proprietary surfaces within the tip by mixing for approximately one minute. Expelling the solution yields purified DNA, the desired product. Diffinity RapidTip (48) for PCR Purification Order #: RT025-048 Qty: 1 Box of 48 tips Your Price: $83 Diffinity RapidTip (96) for PCR Purification Order #: RT025-096 QTY: 1 Box of 96 tips Your Price: $160
Views: 2515 MIDSCI Biotech
PCR Purification Kit
 
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Views: 855 Soumyajit Sen
RapidTips PCR Purification in 60 Seconds (www.midsci.com)
 
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Visit www.midsci.com or call 800-227-9997 to order some today for your lab! RapidTip™ - PCR clean up in a tip in 60 seconds! Purification of PCR products for Sanger Sequencing in just one minute! No bind-wash-elute, enzymes, or magnetic beads necessary! An innovative new technology that removes undesirable impurities from PCR reactions leaving you with nothing but purified DNA for your Sanger sequencing applications. Primers and dNTPs will be gone in 60 seconds. ALL YOU NEED IS: Diffinity RapidTips - pre-packed with our proprietary material and ready to use out of the box! A standard pipettor - single or multi-channel. Microcentrifuge tubes to store purified DNA. 20-30µl PCR reaction volumes Purified PCR product for Sanger sequencing applications. The tip attracts and retains dNTPs, primers and primer dimers of less than 50bp and repels and permits the double stranded DNA targets (such as PCR amplicons) to remain in solution. FEATURES: Excellent Yield: Recovers up to 90% of high quality dsDNA ready to use in subsequent applications. One Minute, One Step: Extremely fast and efficient process to rapidly recover clean DNA. Cost Effective: No capital equipment (e.g., centrifuge, magnetic extractor, vacuum manifold), extra plasticware, or liquids are required. Less labor time required due to simple protocol. Robot Compatible: Easily integrated on automated equipment for high throughput processing and increased productivity. Same purification process, eliminates additional process validation. No Bind-Wash-Elute Steps: Eliminates use of large amounts of reagents and time-consuming, tedious protocols. Effective Purification: Removes up to 90% of primers, ssDNA, and primer dimers. Large Range of Fragment Length: Returns pure DNA fragments of 100bp to 10Kb. Use 20-30µl PCR reaction volumes Easy to Use: Single step protocol requires little to no training of lab personnel. Simple protocol requires no complex operator techniques or interactions with equipment, buffers, or reagents. Environmentally Friendly: Waste is limited to the functional tip. No extra materials, tips, columns, buffers, or reagents required to purify PCR product. Seamless Workflow: Uses standard pipettor so no changes are needed to your current lab workflows. HOW IT WORKS The functional pipette tip will fit on most conventional single or multiple, manual or automated laboratory pipettors. Samples containing DNA and impurities are aspirated into the tip where the impurities are adsorbed onto proprietary surfaces within the tip by mixing for approximately one minute. Expelling the solution yields purified DNA, the desired product. Diffinity RapidTip (48) for PCR Purification Order #: RT025-048 Qty: 1 Box of 48 tips Your Price: $83 Diffinity RapidTip (96) for PCR Purification Order #: RT025-096 QTY: 1 Box of 96 tips Your Price: $160
Views: 864 MIDSCI Biotech
PCR product purification - Part I, cutting a gel
 
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In iGEM-Debrecen 2010 Team we have initiated a project called: "Film in Lab". Our work should guide the beginner lab member, and serve as an open source, for all who have the interest of learning some new lab techniques. The protocols may be found in the 'Open wet ware' account of iGEM-Debrecen Team 2010. These protocols serving as a reference for the "Film in Lab" project. During our work, we had to explain the protocols to other members of the group. Since iGEM-Debrecen Team 2010 is a multidisciplinary group of undergraduates this was essential. Our idea initially was to create an observational way of teaching these techniques, helping the team to progress rapidly. However, the project got lots of sympathy and we have decided to create an open source. Yakir Guri, "Film in Lab" supervisor Balint L. Balint, MD, PhD, Supervisor and instructor of 'iGEM-Debrecen 2010 Team' Members of the iGEM-Debrecen 2010 Team: Beregi Tímea, Erika Berényi, Nagy Katalin, Ozgyn Lilla, Guri Yakir, Keret Ophir, Markovits Daniel, Malka Lior, Shun-Chieh Liu, Katalin Sandor, Kristóf Endre Károly, Bence Daniel. Other members: Alternatív Középiskolai Gimnázium, Budapest (Alternative Secondary school of Economics): Jakab Dorottya Sólyom Alexandra Székely Áron
Views: 3744 debrecenigem2010
Practice 6. Purification of a PCR product from agarose gel 2
 
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Vídeo sobre: Practice 6. Purification of a PCR product from agarose gel 2
Views: 159 andres labastida
How to Prepare Your Samples for DNA Sequencing
 
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Erin Murphy, M.S., a Molecular Biologist with ACGT's DNA Sequencing group, demonstrates how to prepare your samples to ensure high quality DNA sequencing data.
Views: 12744 ACGT, Inc.
Why do we purify the PCR product
 
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Why do we purify the PCR product - Find out more explanation for : 'Why do we purify the PCR product' only from this channel. Information Source: google
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: 25377 Abnova
How to Clean up PCR Reactions with ExoSAP-IT Express Reagent
 
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Learn more or request a sample at http://www.thermofisher.com/exosapit • Five-minute protocol—fast turnaround time • One-tube, one-step PCR cleanup— add reagent directly to PCR product • Novel enzyme technology— irreversibly inactivated in just one minute at 80°C • Complete sample recovery—100% recovery of PCR products, regardless of amplicon length • Eliminates spin columns or beads—significantly decreases time and errors while increasing yield • Compatible—no interference in downstream applications • Scalable—treat a wide range of volumes • Multiple formats—single-tube, 8-tube strip, and 96-well plate formats offer flexibility for manual or automated workflows ExoSAP-IT Express reagents are patented mixtures of exonuclease I combined with shrimp alkaline phosphatase (SAP) in a specially formulated buffer, which remove excess primers and dNTPs following PCR. Exonuclease I removes residual single-stranded primers and any extraneous single-stranded DNA produced during PCR. SAP removes the remaining dNTPs from the PCR mixture that may interfere with subsequent reactions. Adding ExoSAP-IT Express reagent directly to the PCR product eliminates transfer steps to tubes, wells, or columns and conserves amplicons, helping to reduce the chance of cross-contamination; no further processing is needed. After enzymatic cleanup with ExoSAP-IT Express reagent, primers and dNTPs will no longer interfere with DNA sequencing or other downstream applications. Protocol Treat 5 μl of PCR product with 2 μl of ExoSAP-IT Express reagent. Treatment is carried out at 37°C for 4 minutes followed by an incubation period at 80°C for 1 minute to irreversibly inactivate both enzymes. Once enzyme inactivation is complete, your PCR products are ready for downstream applications such as sequencing (Sanger/NGS), fragment analysis, SNP analysis, in vitro transcription, or single base extension
Views: 155261 Thermo Fisher Scientific
DNA Sequencing Purification
 
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My SED 618 video
Views: 1236 pyl1125
MaK Clean PCR purification reagent
 
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MaKClean PCR purification reagent is a simple fast and clean method for PCR purification www.magbiotek.com
Views: 324 magbiotek MBK
Protocol 5 - Prep for Sequencing
 
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Enhance your genetics instruction with The Jackson Laboratory's Teaching the Genome Generation™. FULL PROTOCOL LIST BELOW⬇️️⬇️️⬇️️⬇️ Protocol 1 - DNA Extraction Part 1 - https://www.youtube.com/watch?v=tcPgdR9_t64 Part 2 - https://www.youtube.com/watch?v=1PisbDHKXTU Protocol 2 - PCR Part 1 - https://www.youtube.com/watch?v=QYpX94prb0A Part 2 - https://www.youtube.com/watch?v=MxDgPFNjkbw Protocol 3 - Restriction Digest https://www.youtube.com/watch?v=sEjN_fxJN1s Protocol 4 - Gel Electrophoresis https://www.youtube.com/watch?v=AK8A5OREk34 Protocol 5 - Prep for DNA Sequencing https://www.youtube.com/watch?v=bJfed2B2Pzk Protocol 6 - DNA Sequence Analysis Part 1 - https://www.youtube.com/watch?v=iqAmkNSu3oI Part 2 - https://www.youtube.com/watch?v=7CoIjBvV274 Learn more about Teaching the Genome Generation https://www.jax.org/education-and-learning/high-school-students-and-undergraduates/teaching-the-genome-generation
96 Well PCR Cleanup Kit Video
 
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96 Well PCR Cleanup Kit. 96 Well PCR Cleanup in only 20 minutes. Up to 95% recovery from PCR products and other enzymatic reactions. For more PCR cleanup products please visit: http://www.geneaid.com/ http://www.geneaid.com/products/dna-extraction/pcr-cleanup http://www.geneaid.com/products/pcr-cleanup/pcr-cleanup-kit-96-well Please visit our facebook page: https://www.facebook.com/geneaid/
Views: 232 Geneaid
PCR optimization | PCR technique
 
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PCR optimization | PCR technique - This lecture explains how to optimize PCR reaction for better results. http://www.shomusbiology.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.[15][16] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[8] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, use of disposable plasticware, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA. Addition of reagents, such as formamide, in buffer systems may increase the specificity and yield of PCR.[17] Computer simulations of theoretical PCR results (Electronic PCR) may be performed to assist in primer design. 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: 20351 Shomu's Biology
Agarose Gel Electrophoresis of DNA fragments amplified using PCR
 
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This video is the third lesson in a series of resources detailing the PCR process and surrounding activities. It shows how to analyse a DNA sample using agarose gel electrophoresis, as well as how to make the agarose gel.
Views: 193743 Schools Project
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: 417 New England Biolabs
Ligation of PCR Products
 
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This video is a description of a process called ligation. It's intended to be a pre-lab background before actually performing a ligation. There's also a discussion of the necessity of blunting the PCR product prior to ligation. Ligation of PCR products into plasmid vectors is a critical step prior to transformation. Plasmids that contain inserts will then be used to transform bacteria for cloning.
Views: 23537 Ray Cinti
How to purify microbial and host DNA from stool samples
 
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Learn how to isolate microbial DNA that accurately reflects the diverse microbes in the community sampled. This video will provide an outline of stool microbial DNA isolation, plus some tips and tricks. In addition to stool, the PureLink™ Microbiome DNA Purification Kit can be used to isolate DNA from urine, saliva, swabs, transport media, microbial culture, and soil. The online manual describes the protocol in detail: https://tools.thermofisher.com/content/sfs/manuals/MAN0014266_PureLinkMicrobiome_Stool_UG.pdf For more information on PureLink™ Microbiome DNA Purification Kit, visit: thermofisher.com/microbiome
Diffinity RapidTip for PCR Purification - Demonstration
 
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Product Demonstration Video of Diffinity RapidTip for PCR Purification
Views: 7089 DiffinityGenomics
Ferra-Clear PCR Purification Kit
 
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The Ferra-Clear PCR Purification Kit allows for the purification of DNA from PCR and other enzymatic reactions. Most kits on the market require disposable columns for DNA isolation and therefore are not only restricted to the DNA binding capacity of the column, but also the elution volume required. The Ferra-Clear PCR Purification Kit uses a magnetic nanoparticle solid phase binding system with classic DNA isolation chemistry. Enzymes (i.e., DNA polymerase), primers, dNTPs and salts are efficiently removed. The kit is designed to isolate DNA fragments from 100 bp to 10 kb. The resulting DNA is pure and suitable for a variety of applications including ligation, sequencing, and enzymatic reactions.
Views: 199 Ferradigm
SnoMag™ Gel DNA Purification Kit
 
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SnoMag Gel DNA Purification Kit (http://en.snovabiotech.com/store/index.php?route=product/product&filter_name=gel%20dna&product_id=108) is specially designed for easy and efficient purification of DNA fragments from any agarose gel in either TAE (Tris-acetate/EDTA) or TBE (Tris-borate/EDTA) buffer. SnoMagTM Gel DNA Purification kit comes with Snova proprietary NanoMag® magnetic beads and specially formulated buffer. NanoMag® TC Buffer has been optimized to recover a great quantity of DNA fragments. DNA molecules are adsorbed to NanoMag® TC Buffer in the presence of chaotropic salts, while all non-nucleic acid impurities such as agarose, proteins, salts, and ethidium bromide will be removed during washing steps. The purified DNA will be effectively eluted with 30μl Buffer EL or TE or water. The purified product is tested stable for downstream applications ligation, PCR, sequencing, restriction digestion, or various labeling reactions. http://www.snovabiotech.com
Gel Purifying DNA fragments
 
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How to purify DNA restriction fragments following agarose gel electrophoresis
Views: 5506 Mark Morris
Wizard® SV Gel and PCR Clean-Up System
 
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Vacuum Purification from Gel
GTpure Gel/PCR purification kit procedure 01 -- Binding
 
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This is the video to show the procedure of GTpure Gel/PCR purification kit. GTpure Gel/PCR purification kit offer a fast and easy to highly recovery the pure and high quality of DNA in agarose gel. Please visit our web site http://www.genetechhk.com/ for more detail.
Views: 1292 Genetechhk
The Importance of Sequencing Clean-up  - Seq It Out #9
 
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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 Correctly cleaning up your sequencing reactions is an integral part of the Sanger Sequencing workflow. Without cleanup, you will get suboptimal sequence data. So how does sequencing cleanup work and what tools are available? Let’s find out. To understand the importance of sequencing clean-up, you first have to understand the BigDye Terminator sequencing workflow and the basics of how the sequence information is captured. In short, the DNA that you want to sequence should be isolated and purified. This can be done by a variety of methods. But in general, the cleaner the product, the better the results. Let’s take a look at our lab book Once you have your purified DNA, you can now move on to the BigDye Terminator sequencing reaction. The BigDye Terminator Ready Reaction mix has all of the components necessary for sequencing. Just mix the Ready Reaction Mix, DNA Template, and a single primer and perform your cycle sequencing reaction. For best results, it’s usually better to set up 2 reactions per sample, one using a forward primer and one using a reverse primer. After the BigDye Terminator sequencing reaction, you will need to remove excess labelled nucleotides since these will interfere with the downstream sequence detection process. After the cleanup, the amplicons generated during the sequencing reaction are electrokinetically injected and separated by size, charge, and the dye, which is specific to each of the four nucleotides. The dyes are excited by the instrument laser and detected by the instrument’s camera. If the Sequencing reaction cleanup step is missed or not performed properly, the residual dye in the reaction can out-compete the labelled amplicons for entry into the capillary and can cause reduced signal intensity which can interfere with the instruments ability to make clear base calls. As a result, the data generated will be of poor quality. Several different methods have been developed over the years to cleanup sequencing reactions. Some methods, such as ethanol precipitation, can be labor intensive and time consuming. But there are other methods out there that makes sequencing cleanup easier and faster. The most efficient methods fall into two categories: column and bead based. Column based methods use centrifugal force to pass the solution through a substrate that will bind any unincorporated dyes. Bead based methods use particles in a solution that bind dyes that were not used up in the reaction. Removing the residual dyes from the sequencing reaction gives you much cleaner and more reliable results. Thermo Fisher Scientific offers several different products for sequencing cleanup, such as BigDye Xterminator and Centri-Sep columns. For more information, please visit our website. I hope this video was helpful on Sequencing Cleanup, 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. But remember, when in doubt, just Seq It Out.
PCR purification /BioFACT / PCR cleanup kit / Magnetic bead type / 96well pipettor type
 
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#바이오팩트 #Magnetic bead type # PCR cleanup kit # 96well pipettor # simple / #PCR purification / #96 prep per 1time
Views: 120 손지영
PCR Cleanup Kit Video
 
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PCR Cleanup using the GenepHlow™ Gel/PCR Kit from Geneaid Biotech http://www.geneaid.com/products/pcr-cleanup/pcr-cleanup-kit-genephlow
Views: 635 Geneaid
How to Purify Sequencing Reactions with the BigDye Terminator (BDX) Kit
 
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Learn more at http://www.thermofisher.com/sangerworkflow The Applied Biosystems BigDye Terminator (BDX) Purification Kit provides a fast, simple purification method for DNA sequencing reactions to remove unincorporated BigDye terminators and salts. No more dye blobs! Cleanup is complete in under 40 min and typically requires less than 10 min of labor.
Views: 700639 Thermo Fisher Scientific
ExoSAP-IT Reagent -- enzymatic PCR cleanup with no sample loss
 
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How to perform enzymatic PCR clean up just prior to downstream sequencing, SNP analysis, cloning
Views: 3500 Affymetrix
Small DNA Fragments Extraction Kit
 
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Small DNA Fragments Extraction from PCR product using the Geneaid Small DNA Fragments Extraction Kit. Purify small DNA fragments in as little as 10 minutes! Up to 95% recovery of small DNA fragments. Optimized for 40 bp–200 bp DNA fragment recovery. http://www.geneaid.com/products/gel-extraction/gel-extraction-kit-small-dna-fragments
Views: 472 Geneaid
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: 46523 Abnova
Legionella Aquadien™ Standard DNA Extraction Protocol
 
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For more information, http://bio-rad.com/aquadienstandardDNAextraction In this video, we will demonstrate the proper technique following the standard protocol for filtration of water samples and DNA extraction from the samples for Legionella testing. The AQUADIEN™ Kit allows an optimal DNA extraction and purification from bacteria present in water samples for real-time PCR detection. Visit us on the web to learn more: http://www.bio-rad.com/en-us/product/aquadien-bacterial-dna-extraction-purification-kit We Are Bio-Rad. Our mission: To provide useful, high-quality products and services that advance scientific discovery and improve healthcare. At Bio-Rad, we are united behind this effort. These two objectives are the driving force behind every decision we make, from developing innovative ideas to building global solutions that help solve our customers' greatest challenges. Connect with Bio-Rad Online: Website: http://www.bio-rad.com/ LinkedIn: https://www.linkedin.com/company/1613226/ Facebook: https://www.facebook.com/biorad/ Twitter: https://twitter.com/BioRadLifeSci Instagram: @BioRadLabs Snapchat: @BioRadLabs
Cutting bands out of gel
 
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Learn how to cut DNA bands out of agarose gels.
Views: 14178 labtechniques
BigDye Direct Cycle Sequencing Kit
 
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For more information, please visit: http://www.lifetechnologies.com/bigdyedirect Thanks to the new BigDye Direct Cycle Sequencing Kit it's easy to imagine a simpler sequencing process. Because now, fewer steps and a faster workflow actually yield superior results. Sanger sequencing is the gold standard against which all other sequencing technologies are compared. It's good. Here's a way to make the process even better. With BigDye Direct you can speed and simplify your workflow, and read as close as one base from the primer. You have other things you need to do, and the fewer steps the better. We start with amplifying the template DNA with a PCR step. We will use these same two tubes all through this process. Prepare the PCR using the BigDye Direct PCR Master Mix. The reaction volume is 10 microliters. Place the tubes in a thermal cycler. We recommend the Veriti Thermal Cycler, and perform the PCR in only 64 minutes. Now it's time for the PCR clean-up and cycle sequencing in the same step, add 2 microliters of the BigDye Direct Sequencing Master Mix to both reaction tubes. Then add 1 microliter of the BigDye Direct M13 forward sequencing primer to one reaction tube, and 1 microliter of the BigDye Direct M13 reverse sequencing primer to the other reaction tube. Perform the sequencing reaction, this time placing the tubes in the Veriti Thermal Cycler for only 80 minutes. Now, at this point you may be asking yourself "Wait—didn't they skip a step? What about the PCR clean-up that's normally performed before the sequencing reaction?" Well, BigDye Direct is unique in that the sequencing master mix contains the PCR clean-up reagents AND the sequencing reagents. During the sequencing reaction, the PCR clean-up reagents digest unincorporated PCR primers but do not affect the protected BigDye Direct M13 sequencing primers. So your sequencing reaction can now be performed in optimal conditions. Getting back to the process, once the PCR clean-up/sequencing step is complete it's time to purify the sequencing reactions prior to electrophoresis. Just add the reagents from the BigDye Xterminator purification kit into the reaction tubes and vortex for 20 minutes. The samples are then ready to be analyzed using your Genetic Analyzer System. This is where BigDye Direct offers another significant advantage. When using the POP-7 polymer, the time to read the electropherogram takes place in about half the time, and it also results in higher-quality data. Using the standard process, the first bases of the sequencing are unreadable. They can be read when using BigDye Terminator 1.1 and a POP-6 polymer, but doing so doubles the migration time. However, with BigDye Direct and POP-7, you can easily read the first base after the gene specific primer sequence, allowing you to quickly analyze your sample and achieve better-quality data. Let's take a quick look again at the complete BigDye Direct process. PCR and PCR clean-up, along with sequencing reaction and purification, are all performed in the same reaction tubes, meaning there's less risk of error, and you can easily track your tubes from beginning to end. All in all giving you: • Fewer steps, • A faster workflow, and • Superior results. Imagine a simpler sequencing process. Imagine the BigDye Direct Cycle Sequencing Kit.
T2 PCR trouble shooting no product A.mp4
 
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The second video in the series talks about reasons why no PCR product was obtained. Such reasons include: - some component missing - poor or difficult template - too high annealing temperature
Views: 3046 Matthias Dobbelstein
Watch QIAGEN's unique PCR buffer in action (updated version)
 
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Watch QIAGEN's unique PCR buffer in action: PCR doesn't have to mean tedious and time-consuming optimization. With QIAGEN's unique PCR buffer, you can quickly achieve highly reliable PCR results. Want to find out how? Check out this animation.
Views: 4029 QIAGEN

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