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  1. Plasmid Cloning by PCR

    Type
    Blog Post
    ...important to use a high fidelity polymerase to minimize mutations. The fidelity of the polymerase becomes...as Addgene’s Sequence Analyzer, to allow you to identify which restriction sites are present in a given...compatible with a standard PCR reaction (see PCR Video). Therefore, our Forward Primer will use the sequence... chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need...

  2. Fluorescent Proteins 101: Monitoring Cell Mobility Using Fluorescent Proteins

    Type
    Blog Post
    ...this stage, only cells from the immune system show wide scale mobility with movement from the bone marrow...you can detect signal in thick specimens. See the video below for an example of two-photon intravital microscopy... in diverse tissue environments. These studies provide quantitative measurements of cellular motility,...and skin. Two-photon intravital microscopy. The video above depicts the infiltration of neutrophils expressing...technical specificities have to be taken into consideration especially in the use of fluorescent proteins... Addgene.org Check out Our Fluorescent Protein Guide Pages Find Plasmids for Subcellular Localization...

  3. Improving Transduction Efficiency with Exosome AAVs

    Type
    Blog Post
    ... the widely used 293T. During vector production, assembled AAV vector particles accumulate inside the ...either associated with the outside surface of the exosome or are carried inside the vesicle, which may also...Addgene's viral service Find viral protocols and videos Find AAVs for your research ...

  4. Virtual Science Conference Coming Up? Three Addgenies Share Their Experience

    Type
    Blog Post
    ...physical location didn’t matter. I didn’t have to consider that aspect and I could plan more with regard ...depending on the conference, the audience can help decide which questions get answered. “You can upvote a...presenting is eye contact. “It’s important to keep your video on and look into the camera to make it seem more...on Twitter where each poster presenter had four “slides” to use for their poster, eliminating the need ...to make space for it,” says Bachle. “On the flip side, you can attend conferences for just a little bit...

  5. Crowdfight, a Platform to Boost Scientific Collaboration During COVID-19 and Beyond

    Type
    Blog Post
    ...situation. But a few days later we had an idea. And this idea rapidly grew and became Crowdfight COVID-...modern science in our view. And it was this very idea that research is done better with others than by...has been essential to push certain projects. The idea behind the platform was simple: any person working...remote collaborations Read our Transferable Skills Guide article about external collaborations Resources...

  6. Pushing the Limits of DNA Assembly

    Type
    Blog Post
    ...scarless DNA constructs using homologous regions to guide the joining reaction. BioBrick Assembly will leave...fragments, and generate constructs for producing single-guide RNAs – but it also allows you to skip purification...NEBuilder HiFi so accurate (apart from the high-fidelity polymerase) is the ability to remove, reset and...freely available online. Its simple interface can guide you through both experimental and primer design ...single-stranded synthetic oligo to generate a single-guide RNA (sgRNA). Using only one single-stranded oligo...

  7. Visualizing Translation at the Single Molecule Level

    Type
    Blog Post
    ...Nicholas T., et al. “Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome...sequenced and aligned to the genome to determine genome-wide translational frequency. Although highly useful,...reporter mRNAs. Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that... translated, the SunTag sites on the nascent polypeptide recruit scFV-GFP, generating green puncta that...

  8. The Power Behind NGS Plasmid Validation: seqWell

    Type
    Blog Post
    ...smaller fragments that are then barcoded for identification. This mixture of barcoded fragments is known...for each sample remain highly normalized across a wide range of input DNA concentrations. This process ...of 96 plasmids on a plate) will typically have a wide range of input quantities. This variation is problematic...are produced from large numbers of samples with a wide range of input concentrations (via proprietary chemistry...alternative to Sanger sequencing because the sample identity information is always displayed at the individual...

  9. Cultivating Community Science at BosLab

    Type
    Blog Post
    ...working in academia or industry. At BosLab, we identify ourselves as part of a network of independent ...square, but we saw the potential in the space and decided to reactivate it. We started out by holding a few...from hacking probiotics to filming educational lab videos, while some are looking to develop new skill sets... mind. Thus, to encourage more involvement and provide all our members the opportunity to do molecular...project. This project was inspired by previous city-wide genomic studies and our study will involve getting...

  10. Plasmids 101: NGS Quality Control for Pooled Libraries

    Type
    Blog Post
    ...product because the plasmids in the library are identical except for the insert; there’s no need to sequence...results represent a successful amplification? To provide a point of reference, Addgene commonly prepares...results If your sequencing is successful, your NGS provider should return results in the form of many, many... want! At the end of the day, it’s up to you to decide your threshold of what a good amplification looks...post has helped clarify some of the important considerations when using, amplifying, and verifying pooled...

  11. Plasmids 101: Mammalian Vectors

    Type
    Blog Post
    ...efficiencies and expression levels in the cells. Ideally, the chosen reporter is unique to the cell, is ... generations. Stably transfected cells may be considered an entirely new cell line from that of the original..., NIH3T3, CHO, 293HEK, Jurkat T cells Blocks polypeptide synthesis at 80S; inhibits chain elongation 100...hygB HeLa, NIH3T3, CHO, Jurkat T cells Blocks polypeptide synthesis at 80S; inhibits chain elongation. ... 10-14 days depending on how quickly your cells divide. Gentamicin is often used as a supplement in mammalian...

  12. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ...fall back to? We have outlined some important considerations to help you choose the plasmid prep kit that...or by incorporating recycled paper and plastic. Aside from the more obvious swaps like using smaller bottles...Where can you look for help?  All manufacturers provide specs, protocols, and quality control information...help explain the rationale behind each step and provide troubleshooting advice. In these cases, it can ...between these manufacturers, as price is a large consideration for most labs. Many institutions have preferred...department for these.  While budget is an important consideration, you do want to make sure you’re getting the...

  13. Communicating Your Science With Help From ComSciCon

    Type
    Blog Post
    ...suggests a change to a slide, think about whether the same principle applies to other slides in your deck.  If... craft sentences for abstracts and presentation slides, learn from audiences as they do science outreach...of a new science communication initiative that provides yet more scientists with opportunities to practice...Resources on Addgene.org Check out how Addgene uses videos for science communication ...

  14. Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection

    Type
    Blog Post
    ...in the lab. This is commonly done using calcium chloride which permeabilizes the cell membrane so the bacteria...inject their genetic material into the cell. Once inside, phages can follow one of two different life cycles...both of these life cycles bacterial DNA can be accidentally packaged into the newly created phages. Transfer...referred to as transduction. Transferred DNA once inside the infected bacterium can either exist as transient...Addgene’s protocol page, which has both protocols and videos for techniques in basic molecular biology, plasmid...

  15. Antibodies 101: Monoclonal Antibodies

    Type
    Blog Post
    ...protein. Animals are first injected with a small peptide that represents a short, unique amino acid sequence...isolate individual hybridomas and ELISA is used to identify the hybridomas producing the most specific antibodies...antibody, meaning that the antibodies are largely identical between lots.  Disadvantages of monoclonal antibodies...antibody-encoding genes over time as the hybridoma cells divide. These changes can alter the antibody that is actually...produced by the hybridoma. Any genetic drive can be identified by sequencing the heavy and light chains. To ...

  16. Viral Vectors 101: The AAV Retrograde Serotype

    Type
    Blog Post
    ...Visit Our AAV Guide Page Find Ready-to-use Virus for Your Research Visit Our Lentivirus Guide Page ...understand brain function So far scientists have identified 180 brain regions. Both this geography and the...Using retrograde AAV AAVrg can be used to deliver a wide array of genetic tools for neural interrogation ...where the cell bodies of the projection neurons reside.   Impact for scientists While the mechanisms ...

  17. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    ...of dNTP in the reaction mix, the researcher can decide exactly how far the T4 exonuclease proceeds: at...at the first occurrence of the chosen nucleotide, T4 will switch to polymerizing and then stall due to...the “damage” and producing a complete plasmid. Besides being compatible with any vector, the SLIC protocol...multi-fragment DNA assembly, and its low cost makes it ideal for researchers doing large amounts of cloning. ... 10.1007/978-1-61779-564-0_5. PubMed. JBEI-J5’s guide to SLIC Resources at Addgene Ligation-Independent...

  18. Antibodies 101: Flow Cytometry Controls

    Type
    Blog Post
    ...particular marker, you can use the reference plots provided by the antibody companies or check the literature...sample. The isotype antibody will be virtually identical to the antibody for your marker of interest. It...for that particular marker. This enables you to identify any non-specific binding to know that your positive...unsure what amount of antibody to use, you should consider using an isotype control. Pro tip! Many companies...Cytometry Resources on Addgene.org Addgene's Antibody Guide Browse Addgene's Antibody Collection        ...

  19. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

    Type
    Blog Post
    ...more OptoNBs would allow for more uses both inside and outside cells. References Gil, Agnieszka A., et...exciting because using light to control signaling provides greater spatiotemporal resolution than pharmacological...domain of the Sons of Sevenless (SOS), a guanine nucleotide exchange factor that activates Ras. They expressed...more about optogenetics in Addgene's optogenetics guide ...

  20. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    .... To make a point mutation, you design two oligonucleotide primers with the desired mutation, then use...3-4 phage proteins are needed and these can be provided via a separate plasmid or be inserted into the...insertions and deletions. The figure above shows guidelines for primer design; a desired insertion should...REPLACR-mutagenesis successfully incorporated a 60-nucleotide insertion; beyond this insertion size, primer...introduce mutations. Using commercially available high-fidelity polymerases such as KOD and Phusion, which are...

  21. Protein Tagging with CRISPR/Cas9: A Conversation with Mendenhall and Myers

    Type
    Blog Post
    ... in the human genome. The pair has focused considerable attention on transcription factors, starting...have good antibodies. The results look almost identical. Addgene: What advantages does your system have... also easy to run things in parallel. Addgene: Besides being fast and efficient, what other challenges... Myers: Only a small percentage of the proteins inside a cell have a commercial antibody against them.... to us to get them. Even though we’re happy to provide them, that never works on a large scale. The world...

  22. Oh, The Places You Can Go: Careers in Science Communication - Writing for a Research Institute

    Type
    Blog Post
    ...even come [to grad school]!” He asked if she had considered other careers, which she hadn’t.  He then said... didn’t know if she liked science writing. She decided to start a blog to test it out and discovered she...communications planning.” To beef up her resume, she decided to complete a certificate in Strategic Communications...these difficult pieces: “It’s probably the best.” Besides working through the tough assignments, the skills...important for her work include: “Listening. Evaluating evidence. Critical thinking. Being able to learn things...

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

    Type
    Blog Post
    ...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...variant characteristics they wanted, the team identified Archon1, a novel opsin-based fluorescent voltage...researchers also showed that Archon1 is functional in a wide range of animal models such as mice, C.elegans, ...display technique using small scaffold proteins to identify novel binding proteins that bind to either the...because cPYP binds to BoPD. A similar set of newly identified light- and dark- binder plasmid tools were also...

  24. The Breast Cancer Microenvironment: A Tumor’s Backstage Team

    Type
    Blog Post
    ...tumors may be the stars, there's plenty of action outside of tumor cells, and recent studies (Martins et ...microenvironments made of?  A tumor’s genesis is divided into 3 phases: initiation, progression, and metastasis...to the development and spread of cancer cells. Besides these stromal cell modifications, stromal cells...transportation, exosomes are tools of communication and provide malignancy to the microenvironment itself. They...malignancy, concluding that these cells may be considered "natural tumor suppressors." A growing interest...

  25. Celebrating 15 Years of Scientific Sharing

    Type
    Blog Post
    ...impact.  {% video_player "embed_player" overrideable=False, type='scriptV4', hide_playlist=True, viral_sharing...wonder if providing a viral vector service was a good idea. The scientific community is loving it. Almost 10,000...about this, but definitely not worried. I have confidence that from Business Development to Shipping we...

  26. Uncertainty about Labor Law Brings More Uncertainty to Postdoc Wages

    Type
    Blog Post
    ...to $47,476 per year. This post discusses how a nationwide injunction against the FLSA is affecting universities...injunction against the FLSA updates was granted nationwide. Again, we at Future of Research believe that...higher than the level being raised to now - across a wide consensus of academic stakeholders. However, while...collecting data on the effects of the injunction nationwide and, at exactly one month following the injunction...plans, but have since reversed their position and decided to allow individual departments to go ahead with...

  27. Viral Vectors 101: An Introduction to AAV

    Type
    Blog Post
    ...blogger Didem Goz Ayturk with edits and updates from Addgenies Karen Guerin and Susanna Stroik. Didem Goz ...administered in an organism. These features make AAV ideal for human therapies and in vivo laboratory experiments...therefore the transduced gene is episomal and does not divide and replicate in the same way the host DNA does...Elements Resources on Addgene.org Read Our AAV Guide Find AAV Vectors Browse All Viral Vector Resources...

  28. The time and cost required to make a plasmid

    Type
    Blog Post
    ...thousands of plasmids, using both mass production genome wide screens and the more traditional “one at a time”...time. We also handle the MTA, QC the plasmids, provide helpful resources, and, on the rare occasion things...organized lab saves time and money across the board. Consider alternative cloning strategies There are many ...either don’t scale or have limited scaling. The downside of high-throughput work is that you give up a ...engagement with and use of repositories like Addgene provides large-scale savings. How large? Using Doench’s...

  29. To Each HIS Own

    Type
    Blog Post
    ...affects protein function (Bornhorst 2000).  One downside of polyhistidine tag-based purification, however...contamination, such as mammalian production systems, consider using these more selective substrates along with... containing a polyhistidine tag, you’ll need to decide whether to add the tag to the N- or C-terminus ...the opposite end.  If neither placement works, consider purifying under denaturing conditions. Unlike ...References  Resources on addgene.org Addgene Antibody Guide Ready-to-use antibodies from Addgene More Resources...

  30. Plasmids 101: CcdB - The Toxic Key to Efficient Cloning

    Type
    Blog Post
    ...time-consuming aspects of traditional cloning is the identification of clones that actually contain your insert...enzyme-mediated disruption of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful... insert of interest. Hence correct clones are identified much more efficiently, as those that do not contain...ultimately perform the same function, there is some evidence that ccdB Survival™ can be more difficult to transform...E. coli do not contain the F plasmid (and are considered F-); however, there are a few popular lab strains...

  31. Developing Lab Management Software for Biology

    Type
    Blog Post
    ... of this is gospel, but we think it's worth consideration. Everything will go wrong Whenever an order ... barcodes and containers might not be such a bad idea, tightly coupling them to their contents might leave...easily as needed. In this case, the physical considerations of the laboratory make object composition a...for things will trip everyone up at some point, decide early on what to do in a namespace collision. It...of `dev_name` and `science_name`. You'll need to decide given the technical details of the problem you'...

  32. Career Coaching for Scientists: Why and Where Do I Find One?

    Type
    Blog Post
    ...postdoc offices or professional organizations can provide referrals. There are bad coaches out there, so ...the one for the job. A professional coach will provide an initial consultation meeting or call for free...here are a number I have come across for you to consider. Feel free to add other suggestions in the comments...Scientist. Their motto is “We Turn Academics into Confident and Successful Industry Professionals”. This larger... 6 Steps to Submitting a Resume That Gets Seen  Guide Your PhD with An Individual Development Plan Learn...

  33. Mouse Modeling, Part 2: Breeding and Crossing Mice

    Type
    Blog Post
    ...-lox Read our Genome Engineering Guide Learn more in our CRIPSR guide ...Hemizygous mice can be included in breeding strategies identical to those used for breeding heterozygous mice. ...strains that have been crossed, there are many considerations for effectively breeding your mice. Because...that express Cre but not loxP. With this two-part guide to mouse modeling now complete, you should be prepared...

  34. Open Resources and Plasmid Tools For Studying C. elegans

    Type
    Blog Post
    ... two approaches: single-cell RNA-sequencing to identify neuronal class and deep sequencing of neurons ...database specifically for C. elegans. This database provides detailed information about C. elegans 3’UTRs structures...imaging processing. The combination of these methods provides unique opportunities to develop computational ...self-excise, and covalently ligate their flanking peptides together (Wang et al., 2018).” With the split ...of CRISPR targeting vectors that each contains a guide RNA and repair template of your choosing (Schwartz...

  35. Hot Plasmids - October 2020

    Type
    Blog Post
    ...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ... than the more efficient single-stranded oligodeoxynucleotide donors (ssODN). So how can you use CRISPR...Resources page.  CasPhi is 70 kDa Cas protein identified exclusively in huge bacteriophage genomes. It...dual-targeting hybrid gRNA library can be used to identify genetic interactions between paralogs. The exon-deletion... viral aliquots from our plasmid collection to provide ready-to-use viral preps. Here are some of the ...

  36. Savvy Advocates Needed to Navigate a Scientific Enterprise in Flux

    Type
    Blog Post
    ...across the university. Collective bargaining may provide a better deal for students in areas such as teaching...philosophical landscape of the scientific enterprise will provide challenges and opportunities for us all.  For example...efforts to better account for and track postdocs nationwide. On this level, FoR is currently compiling a ...further increases will be challenging, but will be aided by the development and implementation of new strategies...create tools to better understand human disease.  Outside the lab, he is interested in communicating science...

  37. 5 Tips for Troubleshooting Viral Transductions

    Type
    Blog Post
    ...transduction. Once you’ve harvested, it’s generally a good idea to titer your virus. Getting an accurate count of... blog and be sure to check out our viral vector guide pages to get up-to-date tips for your virus experiments...10325307. PubMed Central PMCID: PMC84922. 5. Ugai, Hideyo, et al. "Stability of a recombinant adenoviral ...immunotherapy." Acute Leukemias VIII. Springer Berlin Heidelberg, 2001. 291-306. PubMed PMID: 10634178. 8. Murnane... on the Addgene.org Check out Our Viral Vector Guide Pages Find Virus Protocols Order Virus ...

  38. Hot Plasmids: Winter 2025

    Type
    Blog Post
    ...resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after herbicide was applied...photostable than fluorescent proteins, making them ideal for single-molecule fluorescence techniques. After...measured pixel and the soma. The initiation site identified in panel (C) is indicated; red line is exponential...research! If they do (or if they don't!), please consider sharing your experience with other researchers...

  39. Addgene's Cancer Collection Pages Connect the Research Community

    Type
    Blog Post
    ...maintain balance – encouraging cells to grow and divide only at the right times and in the right places...trying, as the circulatory and lymph systems are not ideal environments for many cell types. From proteins ... in the development of different cancers and identifying the roles of key proteins involved, with the ...Resources page, investigators may select a pathway and identify any genes within that pathway that are of interest...References 1. Martz, Colin A., et al. "Systematic identification of signaling pathways with potential to confer...

  40. Light Sheet Fluorescence Microscopy

    Type
    Blog Post
    ...multi-view imaging can provide enhanced resolution (8,9).  Thus, LSFM is ideal for 3D reconstruction of...couple-charged device (EMCCD) or complementary metal-oxide semiconductor (CMOS) sensors, it takes only a few...z-stack (8).  One limitation of LSFMs using single side illumination, especially with large tissue, is that...and acquire images of a sample from two opposing sides and merge the images, such as in the Ultramicroscope...
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