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Showing: 481 - 520 of 1100 results

  1. Plasmids 101: Antibiotic Resistance Genes

    Type
    Blog Post
    ...Bleomycin glycopeptide Induces DNA breaks Bactericidal 5-100 ug/mL Erythromycin macrolide Blocks 50S...Resistance to antibiotics is a widely used tool in molecular biology, yet scientists rarely stop to think...when the cells are grown on selective media, and provides those bacteria with a pressure to keep your plasmid...Action*   Working Concentration** Kanamycin aminoglycoside Binds 30S ribosomal subunit; causes mis-translation...mis-translation Bactericidal 50-100 ug/mL Spectinomycin aminoglycoside Binds 30S ribosomal subunit; interrupts protein... Bactericidal 7.5-50 ug/mL Streptomycin aminoglycoside Inhibits initiation of protein synthesis ...Bacteriostatic 50-100 ug/mL in EtOH Polymyxin B polypeptide Alters outer membrane permeability Bactericidal...

  2. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    ...or developmental characteristics that make them ideal for studying in a laboratory environment. We’ve ...manipulation and genetics research. Plasmids are ideal tools for studying genetic modification for their... E. coli biotin ligase, BirA, has been used to identify and study protein-protein interactions in proximity...pathological developments (Vieira et al., 2019) Besides their ability to regenerate, the axolotl has several...several attractive characteristics that make them an ideal model organism; they are relatively simple to maintain...have large, easily modifiable embryos, which is ideal for observing and manipulating the entire developmental...scientific history, highly characterized genome, and widespread utilization, Arabidopsis has made significant...

  3. Antibodies 101: Single Chain Fragment Variables (scFvs)

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    Blog Post
    ...proper formation of the disulfide bond between the VL and VH domains. This bond provides stability and solubility...recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and... two chains are connected by a flexible linker peptide that is usually 15-20 amino acids long and made...lethal to animals.  In the clinic, scFvs’ size also provides advantages over antibodies (Ahmed et al., 2012...oxidizing periplasm of bacteria to allow for proper disulfide bond formation.  expression in strains of bacteria... concentrations in the eye with minimal systemic side effects. scFvs are part of engineered chimeric antigen...determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable...

  4. Harnessing Bacterial Toxins for Allelic Exchange

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    Blog Post
    ...in bacteria, so many that it may be difficult to decide which path to take. A quick and easy way to introduce... the Lambda bacteriophage, is arguably the most widely and productively used  (Thomason et al., 2014)....In recent years, CRISPR-Cas9 technology has also aided in the creation of scarless mutations, both in stand-alone...What are the counter-selectable markers used to identify double crossovers? The most commonly used counter-selection...the genome. Subsequent selection on sucrose will identify double crossovers. This can be an efficient method...unmarked mutants, but it frequently requires considerable optimization to ensure strong counter-selection...Central PMCID: PMC4862005. Jiang, Wenyan, et al. "RNA-guided editing of bacterial genomes using CRISPR-Cas systems...

  5. Hot Plasmids: Summer 2024

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    Blog Post
    ...tissue is identifying the cells you are studying. Antibodies can help reveal cellular identities by targeting...while also supporting imaging, making it easy to identify dozens or hundreds of particles surrounding each... A) Schematic of workflow. B) Detail of spacer peptides (3HB: 11-nm 3-helix bundle; SAH: 60-nm single ...with MagIC-cryo-EM. Nucleosome-like particles identified by Topaz software (green circles), magnetic bead...its chaperone NPM2, but the technique should be widely applicable to other targets. The modular system..., M., Kamath, N., … & Weissman, J. S. (2024). Brainwide silencing of prion protein by AAV-mediated delivery...Samelson, A. J., … & Weissman, J. S. (2021). Genome-wide programmable transcriptional memory by CRISPR-based...

  6. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    ...others. Another peptide-based strategy uses an enzyme to covalently label the peptide with a fluorophore... Balla. "Live cell imaging of phosphoinositides with expressed inositide binding protein domains." Methods...University of Amsterdam). GFP is the most popular, most widely used genetically encoded fluorescent probe. Several...proteins as described above. The smallest tags are peptides that are engineered to bind specifically and with...homology domains can be used to detect specific phosphoinositides (Varnai and Balla, 2008) (Figure 2). RNA production...the continuous effort of numerous scientists world-wide.   Many thanks to our guest blogger, Joachim Goedhart...PubMed PMID: 10748019.  8. Lotze, Jonathan, et al. "Peptide-tags for site-specific protein labelling in vitro...

  7. New Optogenetic Tools for Cytoskeleton and Membrane Control

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    Blog Post
    ...clusters under blue light Check out our Optogenetics Guide to learn more about how these modules work, and ...important components of the cytoskeleton. They provide cells with physical structure, separate chromosomes...microtubules, Opto-katanin will surely be useful for a wide variety of experiments. Opto-RhoGEFs manipulate ...by Rho family signaling pathways. Rho guanine-nucleotide exchange factors (GEFs) activate their partner...system (Mahlandt et al., 2023). These Opto-RhoGEFs provide a reversible and non-invasive way to activate a...efficiency in mammalian cells and in vitro, opening up a wide realm of possibilities. The CRY2-SuperPLD module...membrane or lysosomes (the earlier article also provided CIB constructs for targeting Golgi, ER, and endosome...

  8. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...spectrometry to identify a mass shift proportional to a single phosphate moiety on the peptide of interest...databases of post-translational modifications, identifies a staggering ~250,000 phosphorylation events ...incorporation of phosphoserine (as described below) provides researchers with a valuable tool for understanding...extension and translation of the pSer-incorporated polypeptide. These three components were shown to effectively...mutation. This emphasizes the advantages of bona fide pSer-incorporation into recombinant proteins as ...between different proteins. Beyond these standard considerations, it is important to determine how much phosphorserine...and non-phospho-bands will give the researcher an idea of the purity of pSer-incorporated to non-phosphorylated...

  9. Celebrate Open Data Day with Addgene’s AAV Data Hub!

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    Blog Post
    ...Addgene blog Important Considerations When Using AAVs Three Key Considerations For Precise Neuronal Targeting...your next AAV experiment, use these questions as a guide for designing your future injection experiments....injection and detection methods. For example, you can provide location, titer dose (Example: 1E13 GC/mL) and ...experiment, read our blog post on important considerations when using AAVs. Experimental results: These...Frontiers in Neuroanatomy and summarized some considerations for precise neuronal targeting using AAV on...to give proper attribution.  We hope that this provides a clear overview of what you need to submit data...tracing Resources on Addgene.org Read our AAV guide Browse all  AAV vectors Find ready-to-use AAV preps...

  10. Intro to Our Summer SciComm Series!

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    Blog Post
    ....” Subscribers to Scientific America would be considered the interested public and it would be safe to...scientific or science communication. A Tweet is still considered scientific communication if the language is aimed...communication.  Formal education is education that is provided through a structured environment such as the classroom...will.  Informal education is education that is provided, typically ad hoc, for learners who can choose... only used in the field and terminology that is widely used but has a very specific use-case (definition...the context of disease testing, it can still be considered jargon in most other uses. Many laypeople would...communication work does not explicitly exclude people outside of the original recipients. Instead, it targets...

  11. Multifunctional Neural Fibers: Modulation from Gut to Brain

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    Blog Post
    ...Park et al., 2019). This provided an engineering road map of sorts, giving an idea of the stiffness the fibers...Resources on Addgene.org Optogenetics guide Chemogenetics guide BRAIN initiative plasmid collection ...cumbersome systems during experiments – neither ideal for the intrepid researcher. There existed, therefore...that could continuously monitor multiple systems. Ideally this tool would be wireless, able to monitor outcomes...important as its capabilities are the practical considerations of actually using it - the device has a rechargeable...indeed stable for continuous monitoring. On the gut side of things, the authors optigenetically and chemically...

  12. Fluorescent Protein Travel Awards - FLiPs and Fluorescent Protein Biosensors

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    Blog Post
    ...make her work understandable to people both inside and outside the scientific community. We’re excited to...making their own fluorescent protein technologies widely available to the research community. To honor their... proteins in which Dronpa domains flanked either side of an enzyme’s active site. In these fusions, ultraviolet...domains could be placed in amino acid loops on either side of an enzyme’s active site and still block enzyme... will be able to use similar designs to create a wide variety of enzymes and proteins with inducible activity...post. Fatima Enam: Biosensors for human milk oligosaccharides Fatima Enam is currently a graduate student...fluorescent protein-based biosensors for Human Milk Oligosaccharides (HMOs). HMOs are thought to play an important...

  13. How to Lead a Great Meeting

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    Blog Post
    ...meeting with no idea why you are there. Make the most of everyone’s time by considering the level of meeting...meetings spent on details that do not yet need to be decided. Figure out what you need to accomplish to get .... Team Meetings: For standing team meetings, provide agenda sections for each person to add a bullet...meetings tend to fill the shape of their container. Consider whether you actually need a full hour to achieve...the time down to an hour or less and are able to provide more timely feedback on each other’s projects. ...each of your meeting types (see above). We’ve provided some tips above for what works well at Addgene...FInd Tips on Picking a Research Project Read Other Inside Addgene Posts Resources on the Addgene Website...

  14. Plasmids 101: How to Verify Your Plasmid Using a Restriction Digest Analysis

    Type
    Blog Post
    ...not identical, to their defined recognition sequence. Most enzyme manufacturers make High Fidelity versions...from a colleague down the hall, it is always a good idea to take some time to confirm that you are working...gel correspond to the predicted sizes. Watch this video for a quick overview of how to analyze a restriction...accurately visualize the full size of your construct. Consider buffer and temperature compatibility when digesting...avoid this issue. For your gel: Add ethidium bromide (EtBr) to your gel before pouring it. EtBr binds...your sample settles in the gel well and the dyes provide a visual reference point so you can easily assess...Molecular Biology Reference Find More Protocols and Videos      ...

  15. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...even CRISPR based gene editing. But, how do you decide which tools to use, and what do you need to know...classes of optogenetic tools, and what factors to consider when choosing one.     Figure 1: Example...or inhibit specific neurons without needing to consider secondary signaling pathways. There are many different...signaling pathways. Light exposure removes the block provided by the LOV domain allowing the pathway to be activated...Created with BioRender.com.   Important considerations for using optogenetic tools Once you’ve selected...specific tool. There are many, many options to consider, and the following factors will help you determine...activate or inhibit a cell in succession and maintain fidelity with the stimulus. For example, if you want to...

  16. Antibodies 101: Multiplex Immunofluorescence

    Type
    Blog Post
    ... same considerations apply as for standard IF. However, there are a few specific considerations to keep...standard immunofluorescence, but with some extra considerations (Im et al., 2019; McLaughlin, 2019).    ...your needs, your sample, and targets, you might decide to do simultaneous IF instead (Figure 1B). To label...a good place to get started.  One important consideration when deciding whether you can do simultaneous...suitable antibody against your target protein, consider fusing a protein tag to the target and using an...epitope-based strategy such as Anti-6xHis. You could also consider strategies using other types of affinity reagents...interpret. Check out (Jambor et al., 2021) for more guidelines for creating clear and informative figures. Pro-tip...

  17. Plasmids 101: Restriction Cloning

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    Blog Post
    ...we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples...downstream of the promoter found in the backbone. Ideally, the backbone will contain a variety of restriction...plasmid backbone looks like the one found on the left side of the image below. It has a promoter (blue arrow...single enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert...Methylene Blue Post Visible Light 100 Ethidium Bromide Pre and Post UV Light 0.5 For more information...addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each plasmid to help with...total DNA in a standard ligation reaction. You ideally want a “recipient plasmid : insert ratio” of approximately...

  18. Plasmids 101: Screening Strategies Used in Plasmid Cloning

    Type
    Blog Post
    ...cloning strategy? A screen will help you more easily identify successful clones so you have to weed through ...relies on antibiotic resistance. But what about identifying clones that contain your desired insert? This...ampicillin plates containing X-gal allows you to identify cells that took up the vector and distinguish ...at a few screening methods. Blue-white screen A widely used method of screening is the blue-white screen...example, choose enzymes that only cut once on either side of the expected insert. If the plasmid contains ...planning to go this route, check out our protocol video on using restriction digests to analyze plasmids...Sanger sequencing determines the precise order of nucleotides within a DNA molecule, in this case a plasmid...

  19. Q&A with Karen Katz and Carl Paratore, the Newest Members of the Addgene Board of Directors

    Type
    Blog Post
    ...Addgene provides the scientific community? Katz: The most important resource that Addgene provides is an...about the company and what the goals were and so I decided to join. I’ve been working with Addgene since 2014...distribution of the plasmids and their ability to identify and fulfill a need in terms of helping the scientific...is continuing to look at providing other things besides plasmids to the scientific community through Addgene...perspective, what do you think are important considerations for the future of open science? What about ...exciting about the open science movement is that it provides interesting and unique challenges for intellectual...Addgene blog Learn more about about Addgene in our "Inside Addgene" blog series Subscribe to the Addgene blog...

  20. A Deep Dive into BioBuilder

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    Blog Post
    ...engineering designs and ideas ranged from a biological breathalyzer to pesticide breakdown, production ...engineering of biology. Participating schools are provided with synthetic biology lab kits and lesson plans...recognized name in the field of STEM education worldwide. The BioBuilder curriculum is now taught in almost...and teachers can investigate together. Teachers decide which BioBuilder lessons to teach, and how — some...know-how to design/build/test their own project ideas using synthetic biology.Launching with a kickoff...newsletters, pairs teams with STEM mentors, and provides a nationally recognized platform in March to exhibit...Synthetic Biology Blog Posts Resources on Addgene.org Guide to SynBio Assembly Standards Find Plasmids for Creating...

  21. Clear and Accessible Writing

    Type
    Blog Post
    ...2004, that was a fairly simple goal… and we had no idea how much our understanding of what accessibility...did! From Addgene's very beginning, we had to consider digital accessibility for our website and online... spaces. Alt text is used by screen readers to provide visually impaired visitors with information about...educational content conflicted with plain language guidelines.  Which is why, at the Society for Neuroscience...our workshop how they felt when they read papers outside their field, the answers were primarily negative... Ultracentrifugation First, you’ll want to identify the reader’s goal. Why are they reading this page...and learning how to purify AAV.  Second, you’ll identify your purpose in using that image. Try using the...

  22. Optogenetics + CRISPR, Using Light to Control Genome Editing

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    Blog Post
    ...binds to the target DNA sequence as directed by the guide RNA (gRNA), while the CRY2PHR-activator fusion floats...c-di-GMP in response to far-red light (730 nm). This nucleotide triggers the translocation of the protein BldD...Simply expressing anti-CRISPR proteins does not provide much control over when Cas9 activity is blocked...2020). Fluorescent dCas9 proteins can be used to identify specific genomic loci for imaging techniques. ...architecture. Yu et al., 2020 FAST BphS from R. sphaeroides Split Cas9 Genome editing by NHEJ and HDR. Can...Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing Zhou et al., 2018 ...al., 2016 CRISPR-plus Photocleavable ssDNA oligonucleotides Cas9, photocaged-gRNA Gene editing Manna...

  23. RNA Extraction Without A Kit

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    Blog Post
    ...protocols Find protocol videos on our website Read our Molecular Biology Guide ...material. Then, the homogenate was loaded onto cesium-chloride gradients and ultracentrifuged for up to 20 hours...However, instead of RNA separation using cesium-chloride gradients, water-saturated phenol, sodium acetate... and variations on this no-kit method are still widely used today (Chomcynski and Sacchi, 2006).  View... tissue collection and homogenization. The most widely known of these products is TRIzol® (also called...Allewell and Sarma, 1974). Additionally, ThermoFisher provides a protocol on how to integrate RNAlater® with ... trick is to precipitate the RNA using lithium chloride. LiCl solutions selectively precipitate RNA, but...

  24. CRISPR 101: Cas9 vs. The Other Cas(s)

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    Blog Post
    ...single nucleotide polymorphisms. Fast facts – PAM requirement: none. Best for: high fidelity ssDNA targeting...engineering powerhouse, and for good reason. It is guided by an RNA molecule, the gRNA, to a complementary... and an expanding number of others. Best for: a wide range of DNA edits.   Cas3 – The shredder What ...ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity - sensitive to even a single mismatch in the target...won’t cut it – literally. Cas13 Cas13 is an RNA-guided nuclease, however, unlike Cas9, it targets RNA ...Abudayyeh, O., Omar, et. al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell...Website CRISPR Plasmids and Resources CRISPR Guide  ...

  25. Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening

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    .... "Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library... will introduce you to this new technology, and guide you through the basics of CombiGEM-CRISPR experiments...with which one can design and synthesize CRISPR guide RNAs (gRNAs) for genome editing in large-scale has... has led to the rapid generation of genome-wide gRNA libraries that knock out (Doench et al., 2016; Hart...regulation in biological systems. The CombiGEM platform provides a means to create barcoded gRNA libraries that.... Ma, Hongming, et al. "A CRISPR-based screen identifies genes essential for West-Nile-virus-induced cell...Catch up on Your CRISPR Background with Our CRISPR Guide Pages Find CRISPR Plasmids Find More Tools for Combinatorial...

  26. Plasmids 101: Golden Gate Cloning

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    Blog Post
    ...Addgene’s plasmids are used with a wide variety of restriction enzyme-based cloning methods. Each method...traditional" restriction enzymes in that they cleave outside of their recognition sequence, creating four base...such as BsaI or BbsI), that are located on the outside of the cleavage site. As a result, these sites ...exploits the ability of Type IIS enzymes to cut outside their recognition site and permits DNA fragments...Gate cloning for inserting the appropriate oligonucleotides specifying a gRNA target sequence into a Cas9...amenable to "domestication", you might want to consider using an alternative method like Gateway cloning...cloning or Gibson assembly. Another important consideration is the design of flanking overhangs. Although...

  27. Deep Dive: Statistical Tests (Comparisons)

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    ...to use them, what they can tell you, and how to identify which one is right for you. Statistics – The ...permutation of pairs within the five is different. To identify significant pairings, additional tests, referred...hoc test, done after your initial analysis can provide information on specifically which groups are statistically...within your population? Sometimes this can’t be avoided, but if it can be, you can reduce the family-wise... p value less than or equal to the alpha value divided by the number of null hypotheses. Benjamini uses...uses a complementary approach which identifies a p value cut off. This method entails arranging all calculated... by finding the largest p value where its rank divided by the total number of comparisons performed multiplied...

  28. CRISPR 101: Cytosine and Adenine Base Editors

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    Blog Post
    ...DNA bases without breaking the DNA backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first...hypoxanthine (shown) is the nucleobase component of the nucleoside inosine. Created with BioRender.com. How ...al., 2016). Target-AID acts similarly but not identically to BE3, modifying a 3-5 base window 18 bases ...editors with editing windows as small as 1-2 nucleotides. To reduce off-target effects associated with... created HF-BE3, a base editor containing high fidelity Cas9 variant HF-Cas9 (Rees et al., 2017). HF-BE3...positions 4-7. ABEs 6.3, 7.8, and 7.9 display slightly wider editing windows of position 4-9, although editing...Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their other experiments...

  29. Simple CRISPR-based Epigenetic Editing: dCas9-directed DNA Demethylation

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    ... 2021,  with over 70,000 articles in PubMed, the idea that DNA methylation is linked to disease has now...tools to specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase  When I started...Recently, however, a particular tool has gained widespread popularity – and notoriety – as a tool of choice...   Let me leave you with a few practical considerations: Design your gRNA to overlap the CpG of interest...interest. Having the CpG directly inside the gRNA sequence is a great guarantee that DNMT1 will be blocked...Cas9 Resources at Addgene.org Read our CRISPR Guide to Epigenetics References Charlton J, Jung EJ, ...Galonska C, Charlton J, Mattei AL et al (2018). Genome-wide tracking of dCas9-methyltransferase footprints. ...

  30. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

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    ...sequence or a sequence provided by the depositor. Depositing labs often provide insert sequences or annotated...to check the quality of other services that we provide, including viral preps and pooled libraries. As...this changeover had no impact on our ability to provide requesting scientists high quality data! Addgene's...culture to prepare it for sequencing, we feel confident that these few minor mismatches usually don’t ...differs from what we would expect given the data provided by the depositing lab, we call these “quality ...Sequencing and Analysis Platform to Assess the Identity of Recombinant Adeno-Associated Viral Preparations...

  31. Mouse Modeling, Part 1: Genetically Engineered Mice

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    ... in genetically identical mice. Thus, scientists must genotype each animal to identify the ones with the...-lox Read our Genome Engineering Guide Learn more in our CRIPSR guide ...traits and genetically engineered mouse models provide researchers with useful and adaptable tools to ...background, which means that each mouse is genetically identical and all mice of the strain are homozygous at nearly...inserted gene may have errors introduced to the nucleotide sequence during the DNA repair process. The addition...reporter gene, which replaces the targeted gene and provides a way to track successfully engineered cells. ...

  32. Antibodies 101: Stripping and Reprobing Western Blots

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    ...You’ll want to set up your blot with a polyvinylidene difluoride (PVDF) membrane, which is sturdier and...Western blots are a great tool to identify a protein of interest in a complicated solution like cell ...already run your blot using nitrocellulose, you may decide you have nothing to lose by trying to strip and...abundance of your targets isn’t too different, consider antibody affinity: stronger binding antibodies...matter what stripping methods you throw at it. Ideally, dry your membrane once before any detection steps...(βME) 100–115 mM = 0.7%–0.8% (v/v) Breaks disulfide bonds HCl Adjust pH to 6.7–6.8 Adjust ...addgene.org Western Blot Protocol Western Blot Protocol Video ...

  33. Viral Vectors 101: Chemogenetics

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    ...Chemogenetics has become a popular tool in the field as it provides a noninvasive method for manipulating cell activity... fast-acting optogenetic tools. The advantages provided by chemogenetics are instead that it is noninvasive... crossing the blood-brain barrier.  Another consideration when picking ligands for chemogenetic studies...response you aim to manipulate. It is important to consider that the same receptor can elicit different responses...muscarinic receptor derived DREADDs has been clozapine-N-oxide (CNO). However, concerns have been raised over the...Pharmacokinetic and pharmacodynamic actions of clozapine-N-oxide, clozapine, and compound 21 in DREADD-based chemogenetics... Gomez J, Ramirez M, Hu X, Galvan A, Basu J, Michaelides M, Sternson S (2019) Ultrapotent chemogenetics...

  34. Antibodies 101: Choosing the Right Isotype

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    ...isotype choice. Target and application should be considered The first question you may have is whether a ...monoclonal primary antibody (A). To boost signal, consider using subclass specific secondary antibodies instead...strength (Fig. 1). Monoclonal primaries If you do decide on a monoclonal antibody you may wonder, “Will ...immunoprecipitations. Your sample species should also be considered when choosing an antibody. In some applications...options are available to you, it may be worth considering an antibody raised in a different species.    ...      Table 1: Isotype considerations when choosing antibodies   Complexity is a key factor...with a distinctly conjugated secondary antibody. Consider too that some secondary antibodies can cross react...

  35. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

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    Blog Post
    ..., loci were identified for 61 such diseases. Now single gene alleles have been identified for 4,163, over...surveillance of repetitive gene families and provide a way to guide inheritance that might be less error prone...plasmids, especially those of the CRISPR variety (see slides from my talk below). Largely organized by Dmitry...of items, only a small number of which I could identify. There were dozens of different mushroom varieties...how the NIH sees the future for genomics, and President Obama’s Precision Medicine Initiative and how ...shRNA silencing of one circular RNA they have identified resulted in a cell migration defect. They have...

  36. Magnetic Control of Proteins: More than a Dream

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    Blog Post
    ...magnetoresponsive proteins. Motivated by curiosity Although a wide range of fluorescent proteins and optogenetic tools...possible only in non-biological conditions. So, they decided to search for a magnetic response among other proteins...Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV...imaged area measures approximately 82 mm across. Video courtesy of Andrew York and Maria Ingaramo.   MagLOV...fluorescent proteins.  Currently, the team is working to identify how MagLOV’s structure changes under magnetic ...microscopy tools for biological applications across a wide range of topics. He embraces open science, self-... for?”, and while Andrew and Maria have lots of ideas and are happy to speculate, they know they can’t...

  37. Simplify Cloning with in vivo Assembly

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    ...with low fidelity polymerases and the cost of synthesizing longer oligos, which could be avoided using restriction...into E. coli for propagation. However, while not widely known, plasmid assembly can be achieved in vivo... of a plasmid, order primers that bind at either side of the undesired portion, designed to amplify the... Mutagenesis is performed by designing primers astride the old codon, amplifying the whole vector. The...homology, so performing more complex procedures can be aided by extending homologous sequences (to 25-30 bp)....colonies for simple cloning procedures. Use high fidelity DNA polymerase This will minimize random errors... blog posts Read our blog post on 5 factors to consider before choosing a cloning method Check out all...

  38. Lentiviral Vector Uses and Overview

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    ...envelope have high infectivity in a wide range of cell types, so they’re ideal for work with primary cells or...envelope protein (Env), usually from VSV-G due to wide infectivity, is often used as a substitute for HIV..., used both for targeted modification and genome-wide screening.   Lentiviral vectors in the clinic Since... check out our lentiviral plasmid and biosafety guides before beginning your experiment. Let us know how...PMCID: PMC4273606. 4. Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem...Genome-Scale, Knockout Screening Read about Genome-Wide Screening Using CRISPR/Cas9 Additional Resources...Resources on Addgene.org Check out Our Lentivirus Guide Learn about Virus Biosafety Find Lentiviral Vectors...

  39. Plasmid Preps: Different Purity, Different Quantities, Different Uses

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    ...plasmid from a bacterial culture, it is important to consider your experiment.  It will dictate the amount of...260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus chaotropic agents...agents as guanidine thiocyanate and guanidine hydrochloride used during plasmid extraction. Figure...be entirely endotoxin free. Endotoxin is a lipopolysaccharide of the cell wall of Gram negative bacteria... For these reasons commercially available kits provide extra washing steps to favor the removal of this...want to utilize your plasmid DNA, so that you can decide what will be the optimal type of plasmid preparation...Brush up on molecular biology protocols using our video collection ...

  40. Antibodies 101: Immunoprecipitation

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    Blog Post
    ...When choosing beads, there are several factors to consider. Agarose beads are sponge-like and vary in shape...shape and structure. Their porous surface provides a large area for binding, but antibodies conjugated ...to the capture antibody and is immobilized. The ideal antibody concentration to use will vary between ...antibody is functioning as expected. In order to identify any non-specific interactions, perform parallel... is used to isolate the protein of interest. The wide availability and high specificity of anti-epitope...appealing, but the disadvantages must be carefully considered before using. For example, the tag may affect...routinely use the chromatin IP (ChIP) technique to identify DNA binding proteins involved in histone modification...
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