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We narrowed to 737 results for: TRI

Showing: 61 - 90 of 737 results

  3. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    Published
    March 25, 2025, 1:15 p.m.
    ...filled depends on cellular factors. Multiple labs tried to identify beneficial factors to include in even...bases. Thus, base editors have an activity window restricted to the stretch of ssDNA created by Cas9 binding...

  4. The CRISPR Software Matchmaker: A New Tool for Choosing the Best CRISPR Software for Your Needs

    Type
    Blog Post
    Published
    Nov. 3, 2015, 3:30 p.m.
    ...There is something akin to a frenzy as the crowd tries to weigh and determine the value of every sweet ...This post was contributed by guest blogger Cameron MacPherson at the Institut Pasteur CRISPR software...falciparum (the parasite that causes malaria). I attribute their success to technical expertise, thoughtful...Cas-OFFinder provides a bare-bones data dump of ALL strings in a genome using any IUPAC encoded pattern at ...biotechnology(2014). PubMed PMID: 24880488. 2. Hsu, Patrick D., et al. "DNA targeting specificity of RNA-guided...

  5. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    Published
    Sept. 17, 2024, 1:15 p.m.
    ... antibody manufacturer for wash recommendations. Tris-buffered saline (TBS) or phosphate-buffered saline...abundant protein if you’re stripping, since you’ll lose protein with every strip cycle. Troubleshooting the...Increase volume of wash buffer Incomplete stripping (If stripping/reprobing) Check for antibody signaling...signaling after each round of stripping* Use a harsher stripping buffer Too little signal Large proteins...Western blots can be tricky to get right. If you’ve thoughtfully done the technical design of your blot...need such precise measurements, you can use a colorimetric ladder, visible to the naked eye; a fluorescent... choose from.  Transfer Transfers are a little trickier than gels and may require multiple runs to optimize...

  6. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...e0124958. LOC8-ing mitochondrial fusion with a new tri-chromatic assay Georg Ramm’s lab at Monash University...cumbersome. To overcome many of the limitations intrinsic to DNA-guided systems such as TAL effectors or...modified to use different antibiotic resistances, restriction enzymes and plasmid backbones. pUCXKT contains...gene-specific forward primer containing the desired restriction site (the MCS has several options) and a pUCX-specific...resistance cassette, a ribosome binding site, SacI restriction site and a linker region. The PCR product is ...BMC Molecular Biology. 2013 August 20th; 14(1). TRICK: A method for visualizing the first round of translation...the protein product. The system, cleverly termed TRICK, for Translation RNA Imaging by Coat protein Knock-off...

  7. Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools

    Type
    Blog Post
    Published
    April 23, 2018, 2 p.m.
    ...antibody. TRIM21, an E3 ubiquitin ligase, is the basis of TRIM-away. In normal physiology, TRIM21 recruits...endogenous TRIM21 is sufficient for TRIM-Away, and you only need to deliver the target antibody. TRIM-Away ...Plasmids Podcast! TRIM-Away: Targeted endogenous protein degradation Article contributed by Mary Gearing... proteasome function. TRIM-away compared favorably to drug inhibition, with TRIM-away targeting of mTOR.... To assess TRIM21 toxicity, Clift et al. created stable cell lines overexpressing TRIM21, finding that...changes in expression of TRIM21-associated genes, indicating that TRIM21 overexpression does not aberrantly ... methods for TRIM-Away delivery. For small cell populations, co-microinjection of TRIM21 protein and the...

  8. Plasmids 101: Expressing shRNAs for RNA Interference

    Type
    Blog Post
    Published
    June 17, 2025, 1:15 p.m.
    ...plasmid collection"{% endraw %}{% end_module_attribute %}{% module_attribute "child_css" is_json="true" %}...%}{% raw %}{}{% endraw %}{% end_module_attribute %}{% module_attribute "css" is_json="true" %}{% raw %...padding-right":"180px"}{% endraw %}{% end_module_attribute %}{% module_attribute "definition_id" is_json="true...%}{% raw %}null{% endraw %}{% end_module_attribute %}{% module_attribute "field_types" is_json="true" ...style":"group"}{% endraw %}{% end_module_attribute %}{% module_attribute "label" is_json="true" %}{% raw... raw %}null{% endraw %}{% end_module_attribute %}{% module_attribute "link" is_json="true" %}{% raw %}...user_generated_content":false}{% endraw %}{% end_module_attribute %}{% module_attribute "module_id" is_json="true" %}...

  9. Twenty Years of Sharing: Expanding Accessibility

    Type
    Blog Post
    Published
    Nov. 21, 2024, 10:47 p.m.
    ...journey. Figure 2: Annual distribution of items to countries with a distributor. Arrows indicate the year...amazing 111 countries (Figure 1)! But while we were making the materials available to many countries, import...country to country.   Figure 1: Number of countries Addgene distributes to per year, 2005-2023. We identified... we identified countries that would benefit from a third-party “helper”: a distributor who, among other... India, and Mexico. Table 2: Addgene distributors Distributor Partnership Start Date Country Served...in material distribution after we partnered with them (Figure 2). In others, the distributor helped solve...that we could identify the need for distributors in some countries. ‌We’ve developed systems to allow ...

  10. Evolution of Lab Techniques

    Type
    Blog Post
    Published
    June 21, 2016, 2:30 p.m.
    ...a sequence recognized by the restriction enzyme MstII. When a restriction digest with MstII and southern...than real-time PCR (4). Restriction mapping and modern sequencing Restriction mapping informs on DNA sequence...different restriction enzyme digests. Incubating a DNA sample of unknown sequence with restriction enzymes...has changed, restriction enzymes are certainly not dead yet! Labs still keep restriction enzymes on hand...This post was contributed by guest blogger, Krissy Lyon, a PhD candidate in Neuroscience at Harvard University...three historical techniques: southern blotting, restriction mapping, and sequencing gels, as well as their...fluorescence for fluorescently labeled probes. Colorimetric and luminescence based detection methods are...

  11. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    Published
    March 7, 2023, 2:15 p.m.
    ...categories: parametric and non-parametric. In this blog we will be discussing parametric tests. Parametric tests...be met for a parametric test are as follows:   Parametric test assumptions: Distribution – Normal Variables...   Fig. 2: Examples of data distribution profiles.   Parametric tests  Statistical tests fall into...derived from a theoretical distribution. If your data is normally distributed, it will form (more or less... If your data does not fit parametric assumptions, then a non-parametric test is a better option. Stay...numerical risk allowance in a statistical test. Distribution - spread of data; typically, normal (bell curve...data doesn’t fit the requirements above, a non-parametric test may be the way to go, which we will discuss...

  12. Twenty Years of Addgene Sharing: CRISPR

    Type
    Blog Post
    Published
    April 18, 2024, 1:15 p.m.
    ...top ten countries for each group (Figure 4).    Figure 4: Addgene deposits and distribution by country...deposited by each country. Countries shown are top 10 depositing countries. (B) Total CRISPR plasmids ...requested by each country. Countries shown are the top 10 requesting countries. FR=France; KR=South Korea...in ~40 different countries, and we’ve shipped CRISPR plasmids to labs in ~100 countries.  The impact Addgene...repository has grown to include many CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative...cumulative numbers of CRISPR plasmid distribution and deposits (Figures 1A and 1B); the cumulative number...cumulative number of CRISPR libraries Addgene has distributed (1C), from 2012 to 2023. Addgene’s collection...

  13. CRISPR in the Clinic

    Type
    Blog Post
    Published
    Nov. 2, 2023, 1:15 p.m.
    ...early-phase clinical trials and have shown promising preliminary results (Dimitri et al). CRISPR engineering...editor-based clinical trial for SCD in 2022 and Editas Medicine starting a Cas12-based clinical trial for SCD in ... This ongoing trial is sponsored by Excision Biotherapeutics. Additional ongoing trials The CRISPR therapies...approved or have made significant progress in clinical trials.     Figure 1: Types of delivery and editing...of the cancer-fighting component, CAR. Clinical trials are underway to explore CRISPR’s capacity for both... returning them to the patient. The first human trial involving CRISPR Cas9 for these diseases was sponsored...and CRISPR Therapeutics. Data shared from these trials has been outstanding, with almost all patients ...

  14. Plasmid Cloning by PCR

    Type
    Blog Post
    Published
    March 29, 2016, 2:30 p.m.
    ...assist with restriction enzyme digestion (usually 3-6bp) Restriction Site: Your chosen restriction site for...fusing the upstream restriction site to the forward primer and the downstream restriction site to the reverse...discussed in our restriction cloning post. However, even more than with standard restriction cloning, it is...of a piece of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that...itself are very similar to those outlined in our restriction cloning post with a few quirks specific to the...amplified (usually 18-21bp) When selecting restriction sites, you should use a DNA analysis tool, such...Sequence Analyzer, to allow you to identify which restriction sites are present in a given sequence. You want...

  15. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    Published
    Aug. 2, 2016, 2:30 p.m.
    ...anticipated restriction analysis results (left) and validation PCR followed by Restriction Fragment Length...This post was contributed by guest blogger, Kristian Laursen from Cornell University. Site directed mutagenesis...conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to...the plasmid template (blue), and introduce a restriction site “A*” (in addition to the “A” site already...in the parent vector). Also shown are the “B” restriction sites, which flank the targeted region. Upon ...amplification, the template is removed by DpnI restriction digestion leaving behind only the mutagenized...Polymorphism (RFLP) analysis (right). Introduction of restriction site “A*” results in an additional band when ...

  16. Building and Validating Recombinant Antibodies: A Case Study

    Type
    Blog Post
    Published
    April 5, 2022, 1:20 p.m.
    ... tools created by the Trimmer Lab that will be the first antibodies distributed by Addgene.  The plasmids... constructs. The Trimmer lab found that the recognition sequence for the restriction enzyme BciVI was ...Neuroscience AntiBody Open Resource). The antibodies distributed through NABOR are recombinant antibodies expressed...constructing the plasmids for expression of mAbs, the Trimmer lab isolated RNA from cryopreserved hybridomas,...Constructs were then verified by both diagnostic restriction digest and colony PCR.   Fig. 1: (A) ...can be a threat to future reproducibility. The Trimmer Lab found that in addition to recovering RNA from...lab’s entire hybridoma collection. Instead, the Trimmer Lab moved to direct functional validation by immunofluorescence-immunocytochemistry...

  17. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    Published
    April 11, 2024, 1:15 p.m.
    ..., Vibrio cholerae, Xanthomonas campestris pv. campestris, X. campestris pv. malvacearum, Zymomonas mobilis...plasmid does not have many restriction enzyme sites. Hosts often have a restriction barrier as a defense mechanism...use of their restriction enzymes (Meyer et al. 1977). So if a plasmid has few restriction sites, it may...Methylosinus trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccous denitrificans, Pseudomonas...Shewanella spp., Thiobacillus spp., Xanthomonas campestris IncQ RSF1010, R300B, R1162 Gram-negative...Mycobacterium aurum, M. smegmatis, Paracoccus denitrificans, Pasteurella multocida, Porphyromonas gingivalis...salmonicida, Yersinia enterocolitica, Xanthomonas campestris, X. maltophilia IncW pSa, pR388 Gram-...

  18. Bringing Renewable Energy to Addgene

    Type
    Blog Post
    Published
    Aug. 27, 2019, 1:08 p.m.
    ... of an “electricity aggregation program” for all local households and businesses. Electricity aggregation...Addgenies swapping out printer cartridges can place the spent cartridge in the large cardboard box in ...September all will be powered by 100% renewable electricity! A (not so) shocking transition  In early July...program offers options of between 14% - 100% electricity supplied from renewable sources and we have chosen... As a result, we will be drawing 100% of our electricity from renewable energy projects right here in ...- the majority of which are solar, wind, or hydroelectric in nature. The operation of -80C and -20C freezer...save energy. The transition to 100% renewable electricity will allow us to maintain these particularly ...

  19. MXS Chaining

    Type
    Blog Post
    Published
    Feb. 7, 2017, 3:30 p.m.
    ... mCherry 587nm/ 610 nm human β-Actin Actin 4 Citrine 516nm/ 529nm human α-Tubulin Tubulin Repeatable...principle behind MXS-chaining is repeatable, restriction-enzyme based (chaining-based) cloning. The modules...Modules are assembled one at a time by standard restriction enzyme digestion followed by ligation. Upon ligation...the repeatability of the method. Note that the restriction sites of the MCS are only regenerated at the ... 3’ termini of the ligation product, and the restriction site between the modules is eliminated upon ligation... using these techniques are free of specific restriction enzyme recognition sites. Thus, chaining methods...Technology Pros Cons References Golden Gate Type II restriction enzymes Can assemble up to 10 modules in a single...

  20. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    Published
    Nov. 1, 2018, 12:35 p.m.
    ...unwanted selection markers. Restriction modification systems Because restriction modification systems consist...toxin-antitoxin roles. Restriction modification systems are composed of restriction endonucleases and methyltransferases...preventing the cognate restriction enzyme from cleaving the DNA. A role for restriction endonucleases in plasmid...methyltransferases. Restriction endonucleases cleave DNA at or near specific nucleotide sequences. To ...Escherichia coli in 1995 when introduction of restriction-modification systems onto plasmids allowed cell...these plasmids as compared to those without the restriction modification systems. As with the other toxin-antitoxin...pair is more stable than the other. Here, the restriction endonuclease is more stable than the methyltransferase...

  21. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    Published
    Dec. 10, 2019, 1:58 p.m.
    ...ApoB-LPs through triglyceride/cholesterol does not provide a clear view of either of these metrics (Sniderman...retroviral mouse CRISPR knockout library Article contributed by Alycia Bittner CRISPR pooled libraries are...for measuring ApoB bound lipoproteins Article contributed by Leah Schwiesow A leading cause of death worldwide...ApoB-LPs) are indirectly measured through their triglyceride or cholesterol contents (what is commonly called...called bad cholesterol). However, size distribution and concentration of ApoB-LPs has been shown to have...in vivo. The LipoGlo system represents a new, intriguing approach to expand our understanding of ApoB-...transcription for CRISPR activation in bacteria Article contributed by Will Arnold Baojun Wang’s lab recently developed...

  22. Using Addgene's New Viral Service for Your Research

    Type
    Blog Post
    Published
    Nov. 17, 2016, 3:30 p.m.
    ...accelerate your research.   Why distribute virus? Plasmid distribution is booming, but, over the years... we can help them share and distribute other reagents. Virus distribution came up time and time again ...Addgene! As part of our new Viral Service, we’re distributing lentivirus (with many CRISPR tools included ...with light will be available soon. We’re also distributing AAV preps with particular serotypes that can...here. If you have virus expertise you’d like to contribute to the blog, please email us and we’ll get you...quality control data for each of the materials we distribute. Quality control info can be found on each viral... the Viral Service or any of the materials we distribute, please send an email to [email protected]. Your...

  23. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    Published
    March 14, 2017, 2:30 p.m.
    ...likely to be positively impacted by the trial. During clinical trials, expansion of participants based on ...This post was contributed by guest blogger Subhadra Jayaraman, a doctoral candidate at Binghamton University...surveillance. Dormancy of certain cancers can be attributed to the concept of immunoediting, by way of favorably...TGFβ. T-regs are suppressive immune cells that restrict the production and activity of other effector ...endothelial growth factors (VEGF) in tumors is attributed to a signaling cascade, where lack of oxygen ...therapies include radiation therapy (owing to contributions from Madame Curie), chemotherapy, and surgical...leukemia and lymphoma and are in their clinical trial phase. In all of the above therapeutics, techniques...

  24. FPbase: A new community-editable fluorescent protein database

    Type
    Blog Post
    Published
    May 16, 2018, 1 p.m.
    ...encouraging user contributions (using simple web forms) while also maintaining a relatively strict database design...This post was contributed by guest blogger Talley Lambert, a Research Associate at Harvard Medical School...design of an imaging experiment.  The choice is non-trivial, however, as FPs are tremendously complicated entities...on a web-based interactive visualization of FP attributes (which turned into www.fpvis.org).  That page...and we very much appreciate even those small contributions). Of course, no community-editable database ...simplifies the process of building advanced multi-parametric search queries.  Looking for a bright, monomeric...frequently) for the sake of comparison or ease of retrieval.  These collection pages can be kept private, ...

  25. Visualizing Translation at the Single Molecule Level

    Type
    Blog Post
    Published
    Aug. 1, 2017, 1:15 p.m.
    ...their destination co- or post-translationally. TRICK: Visualizing the first round of translation While... co-opts the physical location of the ribosome, TRICK (Translating RNA Imaging by Coat protein Knock-off...RNA-binding proteins to allow translation to occur. For a TRICK reporter mRNA this displacement leads to a change...RNAs appear red. To enable this color switching, TRICK requires the following components: a GFP protein...translation is necessary for removal of GFP from the TRICK reporter. Delayed translation is known to be important...important in development, and Halstead et al. used TRICK to examine translation of Drosophila patterning ...patterning gene oskar. Early in development, the osk-TRICK reporter was double labeled, indicating translational...

  26. Plasmids 101: Gibson Assembly and Other Long-Homology Based Cloning Methods

    Type
    Blog Post
    Published
    March 1, 2016, 3:30 p.m.
    ...fragments, but uses site specific restriction sites (Type IIS restriction endonucleases) to cleave DNA outside...which have provided appealing alternatives to restriction enzyme cloning. These newer technologies have...They offer many advantages over the traditional restriction enzyme cloning we once relied exclusively on....that does not require the presence of specific restriction sites (or lack thereof) nor a serious time commitment.... If you inserted these two pieces of DNA by restriction enzyme cloning, you would have to do it in two...the smaller overlapping sequences left with a restriction digest. A study in 2013 found that Gibson assembly...PCR step difficult--OR there is no convenient restriction site for linearization. This is a perfect case...

  27. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    Published
    Feb. 27, 2024, 2:15 p.m.
    ...otherwise requested. Distribute pending QC: your plasmids become available for distribution as soon as they...archive any plasmids you've deposited with us and distribute them to scientists worldwide. What's more, depositing... pre-publication plasmids" and set a desired distribution status. You can submit "Published plasmids":...description of the deposit. Select your desired distribution status: Hold for publication: we will initiate... us with publication details or permission to distribute, or Addgene finds your journal publication describing...additional questions. Bacterial Strain Addgene can distribute your bacterial strains if they: are E. coli ... +1 (617)-ADDGENE (233-4363) if you’d like to distribute your bacterial strain(s) through Addgene. Description...

  28. Depositor Week 2020 is Happening Now. Join us!

    Type
    Blog Post
    Published
    Nov. 16, 2020, 2:15 p.m.
    ...that plasmid deposits come from 49 countries* and reached 102 countries. We started from 474 plasmid deposits...deposits coming from two new countries bringing the total to 51 countries with plasmid deposits!  ...Twitter!  That’s today through Friday. There will be trivia, prizes, and more! We wanted to take a week to ...impact in open science. Depositors consistently contribute to our collection so that we can offer the scientific...been requested about 1,500 times from labs in 45 countries*. While the bulk of these plasmid deposits came...you are a depositor or not. We will have Addgene trivia (with prizes!), polls, and shout outs, and we’ll...the schedule for the week: Monday: Fun stats and trivia begins! Tuesday: Depositor features (plasmid deposits...

  29. Hot Plasmids - March 2019 - Anti-CRISPR, 2in1 Cloning, Fluorescent Voltage Indicators, and Photoswitchable Proteins

    Type
    Blog Post
    Published
    March 19, 2019, 1:31 p.m.
    ...Optogenetic control of CRISPR-Cas9 by CASANOVA Article contributed by Cary Valley  Listen to the anti-CRISPR podcast...fluorescent voltage indicator Archon Article contributed by Shreya Vedantam  Listen to the voltage indicator...segment The neurons in the brain compute using electrical impulses to coordinate emotions, thoughts, and... behavior. Historically, scientists study the electrical activity in the brain by inserting electrodes...thousands of proteins for one that can report electrical activity via imaging. They took the light-sensitive...easier with a 2in1 vector cloning system Article contributed by Michelle Cronin  Listen to the 2in1 cloning...pUC57-L3L2) that offer the advantage of using restriction digest cloning to introduce the GOIs into the...

  30. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    Published
    March 8, 2021, 2:41 p.m.
    ...anode/iblot gel matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper...remain intact and the protein is separated through a matrix by charge. In a denaturing western, the protein...smaller molecules moving more quickly through the matrix. For the purpose of this blog, we will be focusing...confirm the size of the protein of interest. An electric current applied to the gel causes the negatively...proteins encounter less resistance in the acrylamide matrix and migrate faster through the gel. For better ... In addition, PVDF membranes can be repeatedly stripped of an antibody and reprobed with different antibodies...sandwiched” together with blotting paper and an electric current is applied causing proteins to migrate...
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