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We narrowed to 387 results for: plasmid dna

Showing: 81 - 120 of 387 results

  1. When Fidelity Matters: A frank discussion about ligase fidelity

    Type
    Blog Post
    Published
    March 6, 2018, 1:49 p.m.
    ...nick-selective DNA ligases like Taq DNA Ligase, HiFi Taq DNA Ligase, and the ATP dependent 9°NTM DNA Ligase are...about it. DNA ligases are enzymes that seal breaks in DNA by joining 5 ́-phosphorylated DNA termini to... several DNA ligases bound to substrate (23, 24). Thermostable DNA ligases, including Taq DNA ligase, ...Blog Learn New Plasmid Cloning Techniques Pushing the Limits of DNA Assembly Plasmid Cloning by PCR ...to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are critical reagents for many ...temperatures (37–75 °C). T4 DNA ligase is highly active in nick ligation and DNA end-joining, but will also...gaps. For example, DNA assembly methods, such as Gibson Assembly® and NEBuilder® HiFi DNA Assembly, require...

  2. Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)

    Type
    Blog Post
    Published
    July 8, 2025, 1:15 p.m.
    ...blog Plasmids 101: Modular Cloning Plasmids 101: Modular Cloning Applications and Kits Plasmids 101: ...through your plasmid stocks. To avoid cross-contamination, we recommend retrieving plasmids from your original... does not equal the number of DNA molecules. A solution of a longer DNA fragment will contain fewer molecules...Calculation for the plasmid volume for MoClo experiments. The term ‘insert’ refers to plasmids carrying each...will find some useful tips to help you make your plasmid construction with MoClo a walk in the park.  Before...promoter, a coding sequence, a terminator, or other plasmid part? Do you want to fuse your protein to any tags... MoClo system allows for directional assembly of DNA sequences, called modules or parts. Assembly is dictated...

  3. Hot Plasmids June 2018 - Reverse transcriptase, nanobody, and protein-DNA interaction tools

    Type
    Blog Post
    Published
    June 27, 2018, 1:16 p.m.
    ...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles ...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need...write about a recent plasmid deposit please sign up here. Listen to the Hot Plasmids Podcast!  MarathonRT... 20 nm of a specific DNA sequence in only 1 min. Using these lentiviral plasmids, catalytically inactive...’s lab at Yale University recently deposited a plasmid for expressing a novel reverse transcriptase (RT...biology and enable the conversion of RNA templates to DNA but suffer from several important shortcomings including...). MarathonRT also faithfully transcribes RNA to DNA with low error rates compared to other RTs. MarathonRT...

  4. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    Published
    Dec. 16, 2015, 3:30 p.m.
    ...evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR is a ground-breaking...that Cas9 cutting efficiency increases when target DNA strand separation is stabilized (2,3). Stable strand...interactions between the groove and the negatively charged DNA and therefore destabilize basal strand separation...cutting. Figure 1: Strand separation and target DNA binding to the Cas9 nuclease. Stable strand separation...sequence independent interactions between Cas9 and DNA, then they could diminish off-target cutting (4)....between Cas9 and the phophate backbone of the target DNA strand via mutations N497A, R661A, Q695A, and Q926A...compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids are available from Addgene here! Future possibilities...

  5. Google Forums Round Up: First Impressions of NgAgo

    Type
    Blog Post
    Published
    Aug. 4, 2016, 2:30 p.m.
    ...target any sequence of DNA. Second, NgAgo target specificity is dictated by a DNA guide sequence rather...(2016). 3. "NgAgo: Genome-editing by a DNA-guided enzyme." DNA-scissors (blog). Blog website. 4. Hsu, ...be produced from a plasmid in cells. Additionally, in vitro assembly of NgAgo/ssDNA requires incubation...genome editing! Check Out Other Argonaute Plasmids from the Doudna Lab Many thanks to our Guest Blogger ...sequencing on select regions of all distributed plasmids as part of quality control, but does not perform...sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease NgAgo! We'll discuss how NgAgo...methods have their own pros and cons (2). The NgAgo DNA guide is inexpensive and can be 5’-phosphorylated...

  6. Plasmids 101: Shuttle Vectors

    Type
    Blog Post
    Published
    May 20, 2025, 1:15 p.m.
    ... Resistance Gene Plasmids 101: Yeast Vectors Plasmids 101: Mammalian Vectors Plasmids 101: The Promoter...other plasmids? If we all use the same molecular code, can I transfect any old bacterial plasmid into ...of replication (ori), where DNA replication begins, so a bacterial plasmid needs a compatible ori that...is transfection. In stable transfection, DNA from the plasmid is incorporated into the host cell’s genome...blog Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection Plasmids 101:...molecular biologist has seen before, showing how DNA sequences are translated into amino acids. And I ...plant-infectious bacteria that can incorporate foreign DNA into plant genomes. Eukaryotes are a bit more complicated...

  7. FastCloning: A Newer, Simpler Cloning Technique

    Type
    Blog Post
    Published
    Nov. 1, 2016, 2:30 p.m.
    ... Blog Plasmid Cloning by PCR Perform Site Directed Mutagenesis by PCR Catch up on Your Plasmid Background...isolation, insertion and amplification of a recombinant DNA without sequence alteration. Molecular cloning techniques... due to excessive PCR cycles or low fidelity Taq DNA polymerase, the construction of specific sequences...be enough time to completely digest the template DNA. Note—templates must have been isolated from methylation...appropriately sized product, purify and sequence the plasmid to confirm no mutations were introduced during ...PCR or any subsequent steps. Using high fidelity DNA polymerases, I have never had any mutations introduced...Amplification efficiency is highest when using high fidelity DNA polymerases like phusion. An 18-cycle PCR leads to...

  8. CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

    Type
    Blog Post
    Published
    Feb. 13, 2018, 2:51 p.m.
    ...This serves to stabilize DNA-protein interactions for subsequent sheering of DNA by sonification. Though...protocols Browse CRISPR plasmids for isolating a genomic locus Browse FRET plasmids for studying protein ...limitations and makes it easier for you to map protein-DNA interaction with limited biological materials. Problems...Though it helps capture weaker or more transient DNA-protein interactions, the reliance on fixation does increase...CUT&RUN, an enzyme-based protocol for identifying DNA binding patterns that overcomes some of the limitations...collected in a single day. CUT&RUN works by using the DNA cutting activity of a Protein A fused micrococcal...micrococcal nuclease (MNase) to specifically isolate DNA that is bound by a protein of interest. First, nuclei ...

  9. Plasmids 101: Origin of Replication

    Type
    Blog Post
    Published
    Nov. 11, 2020, 2:23 p.m.
    ...consider your plasmid's compatibility with one or more other plasmids. Generally speaking, plasmids with the...purify your plasmid DNA: The insert Bacteria tend to maintain fewer copies of plasmids if they contain...elements. The ori is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must...replicons of plasmids are generally different from the those used to replicate the host's chromosomal DNA, but...cell. Figure 1: A plasmid map showing the standard features of a plasmid. So, how do you choose...have only one plasmid type in your cells at a time? Do you want to make a lot of your plasmid? Is the gene...avoid plasmid incompatibility In other words, the best choice of ori depends on how many plasmid copies...

  10. What's New in CRISPR - Winter 2018

    Type
    Blog Post
    Published
    Dec. 18, 2018, 1:17 p.m.
    ...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...CRISPR plasmids to tag human transcription factors with GFP. As part of the Encyclopedia of DNA Elements...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...identified Jennifer Doudna’s lab recently identified Cas14, a family of small DNA-targeting Cas proteins... unlocking) to detect single molecules of RNA or DNA. Now they have developed this further to provide ...Read the paper in Science Find the new SHERLOCK plasmids More precision with dual-nuclease Cas9-Cas9 ...Nature Communications Find the Cas9-Cas9 chimera plasmids A modular toolkit for genome editing in plants...

  11. pSiM24: Simplifying Plant Genetic Engineering

    Type
    Blog Post
    Published
    Sept. 29, 2015, 2:30 p.m.
    ...stability of modified Ti plasmids during plant infection, as well as plasmid yield during preparation ...selectable marker. Left and right T-DNA borders specify the region of DNA that will be transferred to the ...GreenGate Plasmids Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More Plasmids 101: Origin...Agrobacterium carries a “tumor-inducing” or Ti plasmid, which allows it to transfer genetic material into...in two species: Agrobacterium and E. coli. Like plasmids used in other systems, binary vectors are cloned...It’s a lot of information to fit into a single plasmid, so most first-generation binary vectors are quite...pKM24KH.To reduce vector size, they shortened the T-DNA borders to 25 bases each. By switching the E. coli...

  12. Kiran Musunuru on the Newest TALEN Genome-Editing System

    Type
    Blog Post
    Published
    Dec. 10, 2013, 3:13 p.m.
    ...comprise a DNA binding domain and an endonuclease domain. What’s distinctive is that the DNA-binding domain... to target a particular DNA sequence? Musunuru: The kit includes 832 plasmids containing prefabricated...effector nuclease (TALEN) kit consisting of 834 plasmids. The researchers showed they could use their TALEN...and can be programmed to bind to essentially any DNA sequence desired. This technology allows you to create...prefabricated pieces that allow you to build a DNA-binding domain to recognize any desired 15 base pair sequence ...the genome in rapid fashion. The two remaining plasmids in the kit have the rest of the protein in place...fluorescent proteins used to sort the cells after the plasmids have been introduced by transfection or electroporation...

  13. PITChing MMEJ as an Alternative Route for Gene Editing

    Type
    Blog Post
    Published
    Feb. 23, 2016, 3:30 p.m.
    ...repair DNA. The DNA ends are chewed back to reveal homology, allowing the strands to anneal. DNA synthesis...PITCh plasmid to release the cassette, and an insertion-locus-specific gRNA cleaves genomic DNA. These...Guide Pages Find CRISPR Plasmids for Your Experiments Deposit Your New Plasmid Tools with Us  ...Integration into Target Chromosomes). Using their PITCh plasmids, GFP knock-in cell lines can be created in about...Introduction There are three primary methods for repairing DNA after a double-stranded break. HDR copies the sequence... knock-ins, some labs have used NHEJ for whole plasmid integration; however, since NHEJ is error-prone...additional nucleotides flanking the insertion. If the DNA ends anneal incorrectly, MMEJ may also introduce,...

  14. Plasmids 101: Knockout/Knock-In Plasmids

    Type
    Blog Post
    Published
    Dec. 1, 2016, 3:30 p.m.
    ...disrupting it with an piece of DNA designed in the lab. Specially constructed plasmids can be used to replace ...gene. Here, we'll describe the techniques and the plasmids used to inactivate specific genes in mammalian...mind when choosing a knockout strategy! Knockout plasmids Homologous recombination is a mechanism to accurately...exchanged between two similar or identical molecules of DNA. Gene targeting takes advantage of this natural process...randomly integrated all or large portions of the plasmid. Deliver your construct to your target cells. After...Cre-mediated recombination.   Knockout/Knock-in plasmids Gene targeting methods also make it possible to... and insert GFP into the genome, we’d create a plasmid similar to the one shown below, where the sequence...

  15. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    Published
    Oct. 6, 2015, 2:30 p.m.
    ...samples at Addgene - DNA sent from depositors, bacterial glycerol stocks, prepped DNA ready for our quality...bacteria with the plasmid can grow. As the bacteria divide, they replicate and retain the plasmid. This allows... Inside Addgene Posts! Learn all about Plasmids in our Plasmids 101 series! ...Every plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the...dishes, but close to 80% of samples are received as DNA in a microcentrifuge tube. Once a sample enters Addgene...about Developing Lab Management Software    The plasmid's journey begins First, each package is opened and... to hundreds of samples, with an average of 10 plasmids per deposit. Each week an average of 16 deposits...

  16. Easi-CRISPR: Generating Knock-In and Conditional Mouse Models

    Type
    Blog Post
    Published
    April 5, 2018, 12:42 p.m.
    ...Wenning Qin. Single-stranded DNA repair templates Short single-stranded DNA templates have been shown to...editing. How to use Addgene plasmids for Easi-CRISPR You can use Addgene plasmids to create a repair template...of Addgene plasmid options for templates - see the example at right using blue flame plasmid pmScarlet_...desired insert (e.g., mScarlet).   Plasmid ID Plasmid Name Tags 69090 pMJ915 MBP (N ...out our CRISPR Guide Find Plasmids for Cas9 Purification Find CRISPR Plasmids for Mammalian Expression ...pmScarlet_C1! After creating the T7-DNA template, you'd use ivTRT to create the ssDNA construct as described in ...pre-CRISPR was very arduous. First, you’d construct a plasmid with your desired changes, long homology arms and...

  17. Plasmids 101: Blue-white Screening

    Type
    Blog Post
    Published
    June 4, 2015, 1:03 p.m.
    ...This post is part of our ongoing Plasmids 101 series. Plasmids 101 will provide you with an overview ...of the Plasmids 101 posts here. Now that we have covered antibiotic selection here at Plasmids 101, we...α-complementation plasmid. This way you can quickly and easily identify colonies that not only have your plasmid (antibiotic...rescued by genes supplied on the transformed plasmid. Such plasmids may contain genes that allow for the use... able to select for colonies that contain your plasmid is a great start when cloning, but how about being...being able to choose those that contain plasmid with an insert? Blue-white selection is a widely used ...far, so good, but how does this help us screen plasmids? Blue-white screening in the lab Scientists discovered...

  18. Nanoblades: Tiny CRISPR Ninjas for Genome Editing Difficult Cells

    Type
    Blog Post
    Published
    Sept. 26, 2019, 12:50 p.m.
    ...effects when compared to DNA transfection of gRNA and Cas9 expression plasmids. These results are in line...infiltrate cells. Options for CRISPR delivery include plasmid transfection, RNP electroporation, and viral transduction...Nanoblades because they think of these particles as tiny DNA cutting ninjas that deliver CRISPR to multiple types... Gag in place of SpCas9. gRNA A gRNA targeting a DNA sequence of interest is expressed in 293T cells and...types they tested. Interested in using Nanoblades? Plasmids are available from Addgene. Genome editing primary...generally having lower off-target edits compared to DNA transfection.  Homology directed repair (HDR) with...-based HDR allows for the precise insertion of a DNA sequence ranging from a single nucleotide change ...

  19. Evolution of Lab Techniques

    Type
    Blog Post
    Published
    June 21, 2016, 2:30 p.m.
    ...without sickle cell anemia, two DNA fragments are observed because the DNA has the site where MstII cuts... cell anemia the enzyme does not cut the DNA so only one DNA fragment is observed. Today Southern blotting...detects and quantifies a gene or DNA sequence of interest by recording DNA abundance throughout the amplification...mapping informs on DNA sequence through the use of restriction enzymes that cut DNA at specific sites ...specific fragments of a DNA sequence of interest to create a physical map of the DNA. Starting in the 1970s...innovation is nanopore-based DNA sequencing which reads the nucleotides of a DNA strand as it slips through...technologies nanopore-based DNA sequencing machines are relatively small meaning that DNA sequencing could move...

  20. Hot Plasmids Podcast Episode 3: Optogenetics Tools, Improved CRISPR Base Editors, & More!

    Type
    Blog Post
    Published
    Oct. 25, 2017, 1 p.m.
    ..., DIY DNA ladders. You can find additional hot plasmids in our newsletter or on our hot plasmids webpage...Improved CRISPR Base Editors Hot Plasmid 4: pPSU Plasmids for generating DNA molecular weight markers.  Listen...webpage.   Hot plasmids covered in this segment Hot Plasmid 1: Photocleaveable PhoCl protein for controlling...In the third episode of our Hot Plasmids podcast series, you'll learn about optogenetics tools for controlling.... Listen to Our Segement on PhoCl Segment  Hot Plasmid 2: Ouabain selection for increased CRISPR editing...Listen to the Segement on Ouabain Selection  Hot Plasmid 3: CRISPR base editors with enhanced range and ...Listen to the Segement on the pPSU Plasmids Special thanks to our sound engineer/producer, Eddy Page!...

  21. 15 Hot Plasmids from 2017

    Type
    Blog Post
    Published
    Jan. 9, 2018, 3:02 p.m.
    ...created DNA ladders (aka DNA molecular weight markers) that can be used to measure the size of DNA fragments...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief articles...articles provide a synopsis of a plasmid or group of plasmids' functions and applications. We hope that... you to find and use the plasmids you need. You can find all the hot plasmids from 2017 below. With over...over 50,000 plasmids, we can't write posts for every great plasmid that comes into the repository, but...write about your plasmids in a future blog post. No time to read? Listen to our hot plasmids segment on the...editing for precise genome modification. Plasmid ID Plasmid Name Cas9 Variant Speacial Features 85169...

  22. Hot Plasmids and Viral Preps - September 2021

    Type
    Blog Post
    Published
    Sept. 21, 2021, 1:15 p.m.
    ...subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These articles...recent plasmid deposits and we hope they'll make it easier for you to find and use the plasmids you need... CRISPR plasmids are always being added to the repository. To find all of the CRISPR plasmids available...known or suspected to be involved in DNA damage response and DNA repair.  A new CRISPR pooled library ...only required three components: a master mix, the DNA of interest, and cell extract.    Graphical...They allow single-nucleotide modifications to edit DNA without requiring a double-stranded break. The Xiao...Xiao Wang Lab has recently deposited a set of plasmids that helps identify base edited cell populations...

  23. Plasmids 101: Screening Strategies Used in Plasmid Cloning

    Type
    Blog Post
    Published
    Sept. 15, 2020, 1:15 p.m.
    ...once in the plasmid backbone and another enzyme that cuts within the insert. If the plasmid contains the...or absence of DNA using lysed bacterial colonies, without the need to prep your plasmid. In this case,... PCR product if the plasmid contains the insert and no PCR product if the plasmid does not contain the...precise order of nucleotides within a DNA molecule, in this case a plasmid. Sequencing is one of the surest...resources on the Addgene blog Get all the plasmid basics in our Plasmids 101 series Find more molecular biology...If you’re cloning a plasmid, you’ll need a way to find the needle in the haystack: the one perfect clone...clone that contains the plasmid you’re looking for out of the many cells that don’t. One way to begin ...

  24. Lab Automation at Addgene

    Type
    Blog Post
    Published
    March 22, 2018, 1:28 p.m.
    ...scientists share plasmids across the globe, we’re working on ways to send plasmids as DNA as opposed to ...rate! Automating DNA purification We're developing ways to prep large quantities of DNA because some countries...more efficient. Here, I’ll discuss transformation, DNA purification, and filling vials with agar. I’ll also... transformation. With our ever growing rate of plasmid deposits (over 100 deposits per month!), manual...Biomek. The FXP id also able to do high throughput DNA preps and other molecular work if we need it to. ... shipments, we’ve needed to find a way to purify DNA from many bacterial samples at the same time as opposed...Qiagen BIOROBOT Universal Platform and it suits our plasmid prepping needs perfectly. Working with Qiagen, ...

  25. Plasmids 101: 5 factors to help you choose the right cloning method

    Type
    Blog Post
    Published
    Aug. 21, 2018, 12:31 p.m.
    ... Read other Plasmids 101 blog posts New to plasmids? Read our article on What is a Plasmid? Download the...cloning different types of DNA, such as cDNA, PCR fragments, or genomic DNA. Also remember that commercially... the Plasmids 101 eBook Resources on Addgene.org0 Check out protocols for plasmid cloning Find vector... methods are more amenable to combining multiple DNA fragments than others. In today’s post, we will focus...technique, you first perform a round of PCR to generate DNA fragments with 15-40 bp of homology at their ends...combined with an exonuclease, a polymerase, and a DNA ligase to carry out a pooled set of ligations in ...enzymes, optimize reagent concentration, or purify DNA fragments from gels. If you’re relatively new to ...

  26. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    Published
    June 14, 2018, 11:44 a.m.
    ...genome. The genome itself is a linear double-stranded DNA of ~36kb, with inverted terminal repeats (ITR) present...fiber, penton and hexon proteins. The viral genome DNA is further protected by histone-like proteins forming...regions and can accommodate up to 8.2 kb of transgene DNA. The second generation vectors are deficient in E1...down into 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular...adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene of interest...how to generate the final recombinant adenoviral plasmid construct, please see Addgene’s Adenoviral Guide...2.*Pro-Tip*: once the correct recombinant pAdV plasmid is identified, re-transform into a bacterial strain...

  27. Plasmids 101: The Wide World of Natural Plasmids

    Type
    Blog Post
    Published
    Oct. 8, 2024, 1:15 p.m.
    ...toolbox. Natural plasmids, also called environmental plasmids, are all around us! A plasmid is a small piece...colicin-producing plasmids have been referred to as “Col plasmids” or “Col factors.” Virulence plasmids: microbial...Old Solution but a New Problem Plasmids 101: What is a Plasmid? Plasmids 101: Transformation, Transduction...F. (2023). Plasmid classifications. Plasmid, 126, 102684. https://doi.org/10.1016/j.plasmid.2023.102684...when we think of plasmids, both Archaea and Eukaryota can also harbor these circular DNA passengers. Most...., 2001) Beneficial traits of natural plasmids Natural plasmids often convey a variety of functions that...functions of natural plasmids. Created with BioRender.com. Resistance (R) plasmids: microbial defense...

  28. CASTing Off for New Shores in Human Genome Editing

    Type
    Blog Post
    Published
    June 10, 2025, 1:15 p.m.
    ...and was able to integrate DNA payloads of up to 15 kb. Find evoCAST plasmids here! The new horizons of...proteins integrate a payload sequence into genomic DNA downstream from the Cas target. Conveniently, the... (Tns) to integrate large sequences into genomic DNA downstream of gRNA-specified sites. Created with ...moved a gene required for phage propagation to a plasmid that lacked a promoter — only CAST integration ...20 mutations in TnsB, the subunit that catalyzes DNA insertion, along with both evolved and engineered...Transposon-encoded CRISPR-Cas systems direct RNA-guided DNA integration. Nature, 571(7764), 219–225. https://... Engineering Using CRISPR-Transposons Degrading DNA with Cascade-Cas3 Typing CRISPR Systems Prime Editing...

  29. A Look at Addgene's QC Process

    Type
    Blog Post
    Published
    May 6, 2025, 1:15 p.m.
    ...blog Addgene's Tips for Plasmid Quality Control Plasmids 101: In-house NGS Plasmid Quality Control Now at...tools that rely on DNA amplification and manipulation to produce and edit plasmids. These are prone to...sequencing all incoming plasmids. If you are interested in requesting one of these plasmids, you can request ...data for each of their plasmids, from descriptions of key components of the plasmids to partial, full, or...to ensure all plasmids pass our high-quality standards and contain the expected plasmid features.  ...Once Addgene receives deposited plasmids, they undergo complete plasmid sequencing using an in-house Illumina...will compare the NGS results to the plasmid information, other plasmids in the deposit, NCBI sequence and...

  30. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    Published
    Dec. 22, 2022, 2:15 p.m.
    ... frequent DNA repair mechanism in general. On average, it accounts for less than 10% of DNA repair, with...though exogenous DNA can be used as well. Below are some considerations for designing donor DNA for successful...homology-directed repair pathway, designing the best donor DNA, and avoiding common mishaps in this class of genome...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. ...knock-ins. Knock-in mutations typically require an exact DNA sequence to be incorporated at a precise genomic ...which is done through the design and use of a donor DNA molecule. Your donor molecule will need to have homology...This homology will guide the cell to use the donor DNA as a template for repair, which will result in the...

  31. Illuminating Epigenetics with A FRET Based Biosensor

    Type
    Blog Post
    Published
    Nov. 19, 2015, 3:30 p.m.
    ...Chromatin is a complex of DNA and histone proteins that compacts cellular DNA into chromosomes and reduces...reduces the risk of dangerous DNA damage. If the way DNA is wrapped around histones changes, then gene expression...methyl- groups to the DNA, which mostly occurs at CpG islands (regions of the DNA with a high frequency...gene expression that do not involve any changes to DNA sequence (Epigenetics literally means ‘on top of ...This is how most cells in the body contain the same DNA but have very different structure and function, and...change as well. The more frequently and tightly bound DNA is to histones, the more compacted it becomes. This...associated with reduced life-span, but no changes in the DNA sequence could be found. Angelman syndrome and Prader–Willi...

  32. Viral Production at Addgene

    Type
    Blog Post
    Published
    Oct. 24, 2023, 1:15 p.m.
    ...the transfer plasmid, a plasmid encoding rep and serotype-specific cap genes, and a plasmid encoding adenoviral...transfer plasmid Addgene uses a rigorous barcode matching system that follows the sample from the DNA tube.... coli. In addition, plasmids are typically prepared using endotoxin-free plasmid purification kits.   ...confirmation of transfer plasmids used in Addgene’s viral production. Barcoding allows our DNA and virus to be ...of the viral genome and the absence of unexpected DNA contaminants. The data is also used to determine ...contamination is minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic...lentiviral vector, incubated for 72 hours, and genomic DNA is extracted. Primers and probes targeting integrated...

  33. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    Published
    April 4, 2019, 12:53 p.m.
    ...Mobile-CRISPRi ICE element forms a conjugative plasmid-like DNA circle and is transferred to recipient bacteria...Browse our CRISPR interference plasmids Find bacterial CRISPR plasmids Read our CRISPR guide ... into the genome and is propagated during normal DNA replication and cell division. Mobile-CRISPRi does...transposon system. This system has two parts: a plasmid expressing the Tn7 transposition genes (tnsA, tnsB... tnsB, tnsC, tnsD, and tnsE), and a plasmid that contains the Mobile CRISPRi cassette flanked by left ...copy of the RP4 transfer machinery mobilize these plasmids for transfer to recipient cells during a tri-parental...due to site selection by TnsD, a sequence-specific DNA binding protein. Selection removes donor bacteria...

  34. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    Published
    March 7, 2017, 3:30 p.m.
    ...primers flanking my edits in genomic DNA and doing PCR on genomic DNA. The surveyor assay itself was relatively...BRDN0000561167) (Plasmid #80034) and BRAF gRNAs (BRDN0001145345 and BRDN0001146266) (Plasmid #76479 and #76480...that Surveyor nuclease cleaves double stranded DNA (dsDNA) at basepair mismatches. To use this tool to ... denature and renature the dsDNA. Upon renaturation, you will generate DNA duplexes with one strand from...viruses, but you can find a list of validated gRNA plasmids on our website. There are a few reasons to target...gRNA-transduced Cas9-A549 cells are used as a control. DNA was analyzed by PAGE. The basis of the Surveyor...these mismatches are indicated by the presence of DNA cleavage products. The appearance of the 428 bp band...

  35. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ...reflection of the large size of the plasmid or silencing of the plasmid by DNA methylation. Another downside...into cells as a donor plasmid containing the transposon, along with a helper plasmid that expresses the ...reprogramming factors. Non-replicating Plasmids: Generating iPSCs with plasmid-based expression requires serial... generating iPSCs by plasmid transfection where 2 of 11 clones tested had plasmid integration. Another...the dose of plasmid the cells receive over the whole reprogramming period and the plasmid will get diluted...larger plasmids have lower rates of transfection. Minicircles: Minicircles are like mini-plasmids. They...periods of time than traditional plasmids due to lower activation of DNA-silencing mechanisms. While minicircles...

  36. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    Published
    Oct. 11, 2016, 2:30 p.m.
    ... consists of a Ti plasmid, into which the GOI is inserted, and a Ti "helper" plasmid, which contains Vir...necessary for T-DNA processing and insertion into the plant genome. B) The Ti plasmid and helper are transformed... to plant cells. The region of the Ti plasmid between the T-DNA borders (yellow) are recognized by the...for creating plant expression plasmids, including plant CRISPR plasmids from the Yiping Qi lab and the...plant-specific modifications to commonly used CRISPR plasmids are necessary to use the CRISPR system in plant...et al., 2013). Addgene carries Cas9-containing plasmids for knockout, activation and repression of target...expression of both Cas9 and the gRNA off of the same plasmid. Delivering CRISPR components to plant cells Once...

  37. What's New in CRISPR - September 2019

    Type
    Blog Post
    Published
    Sept. 10, 2019, 12:56 p.m.
    ...CRISPR plasmids available at Addgene. We will still periodically focus on specific CRISPR plasmid tools...scaffold and a domain needed for DNA unwinding. They’ve deposited plasmids for active and inactive CasX for...CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ...Jennifer Doudna’s lab discovered a new Cas protein, CasX, from metagenomic analysis of microbial DNA from ...In this system, Cas9 generates a double-stranded DNA break at the genomic loci of interest and on either...vector sequence. Homology arms that flank the donor plasmid cargo are complementary to either side of the double-stranded...expression. Read the paper in Nature Find the CasX plasmids Drug inducible transcriptional activation and...

  38. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    Published
    May 24, 2018, 1:20 p.m.
    ...complementary DNA (cDNA), and then use PCR and molecular cloning to insert the cDNA into the desired plasmid backbone...experiments in planarians with their recent plasmid deposit. These plasmids enable two main types of experiments... cloned into plasmids to investigate for each of these planarian species. These plasmids all have a PR-T4...dsRNA. Students can produce DNA templates via PCR off of the same plasmid and use these to create riboprobes...off of linearized DNA templates which they can easily generate off of a circular plasmid via PCR. Typically...Resources at Addgene.org Find plasmids from the Sánchez lab Find plasmids for other developmental models...of specific gene expression. The 49 deposited plasmids enable students to study gene expression and functions...

  39. A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells

    Type
    Blog Post
    Published
    May 7, 2020, 1:15 p.m.
    ...to the genomic DNA of interest  Figure 1. When ABEs are in action they bind DNA so that the TadA...Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551:464–471 . https://doi....TadA domain can edit the short single-stranded DNA stretch that is accessible. Image from Gaudelli et al...in 40 new ABE8 variants. When comparing on-target DNA editing efficiency in a human cell line for these...ABE8.x-m/d constructs demonstrated a 2.1x on-target DNA-editing efficiency in a human cell line while reducing...pyogenes Cas9 requires the target site in the genomic DNA to contain a specific protospacer adjacent motif ...followed by 2 guanines). This limits the use of ABEs as DNA sites that do not contain a suitable PAM cannot be...

  40. A History of Genome Engineering in Popular Culture

    Type
    Blog Post
    Published
    Feb. 25, 2020, 2:15 p.m.
    ...of the DNA. Finally, in 1953, Franklin along with James Watson and Francis Crick resolved the DNA double...letters of the DNA get translated into the twenty amino acids. Once scientists established that DNA encoded ...dinosaurs by extracting the DNA from fossilized amber and supplementing it with the DNA of present-day frogs ...enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain (FokI). Zinc finger nucleases...designed in 2005. The DNA-binding domain can be engineered to target specific DNA sequences while FokI ...TALENs) were engineered. TALENs follow a DNA-binding and DNA-cleaving strategy similar to the ZFNs, but...mutations, delete short stretches of DNA and even insert new DNA at the target genomic loci.  A last ...
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