...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...herpes simplex virus to facilitate efficient replication. The most common production system, however, ...RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid genes.  ...al., 2010). For certain in vitro and in vivo applications, you can consider using unpurified AAV from ..., 2017). You can learn more about common AAV applications by reading our Viral Vectors 101 series!  This...

...activities with the public, so I reached out to people who were educating the public and organizing events...wanted a career in science outreach: engaging the public to promote an awareness and understanding of science...for researchers interested in connecting with the public. My experiences in grad school, though often called...outreach, not only can I easily share science with the public, but I can watch and learn from the experts in ...

...well as suitable applications. Expression System Components of System Application Mammalian expression...CRISPR mammalian expression systems, the typical applications for each, and potential delivery methods.   ...Select the appropriate CRISPR tools for your application: Wildtype Cas9 or Cas9 nickase are appropriate...or more sgRNAs and Cas9 are required for any application. If you want to include a homology directed repair...

... far beyond the laboratory and seriously damage public trust in scientists, experiments involving potential...strategies is strongly advised. The effects are multiplicative, and there are very few gene drive experiments...species in question), why not use them whenever applicable?Against this, consider the cost of an accidental...An accidental release could delay real-world applications against scourges such as malaria and dengue ...about gene drives can be found in the referenced publications. Thank you to our guest blogger! An inventor...

...plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., Lee, C., Pleiner...alternative method for immunodetection? Enter the Görlich lab and their anti-mouse and -rabbit IgG secondary..., Alexa Fluor 568, or Alexa Fluor 647 before application to the sample. Staining with this multi-color...available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox of anti–mouse and anti–rabbit...

...outside of your organization Public Speaking Time management Managing a team Conflict resolution Organization...not-so-technical skills you’ve built over time are only applicable to those positions you have already held. Imagine...have the desired degree, you have mastered the applications they use, you’ve gained experience working with...project, one can assume that you’ve had to flex your conflict management and communication skills quite a bit...

...develop diverse fluorescent reporters is a useful application of high-throughput cloning. MXS Chaining is one...plasmids for fluorescence imaging or flow cytometry applications in mammalian cells. The modules used in this...transgene expression, or a variety of other clever applications. In one example, modular constructs generated...methods are suited for particular downstream applications. The tradeoffs between various cloning methods...technique to engineer various constructs with applications in flow cytometry approaches in mammalian cell...

...detection of the product. As an added benefit, replicates are built into the technology. One well (containing... 2016). This is particularly important if your amplicon is longer than 150 base pairs. While we use ddPCR... ddPCR for AAV titration, there are many applications for the technology.  Droplet digital PCR is well...being used in the detection of microbes. Some applications include measuring viable probiotics and detecting...detecting circulating pathogens.  With its many applications and ease of use, droplet digital PCR is becoming...

...retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, we will mostly use the gamma-retroviruses...wild-type viral particle — and of the normal, replicative life cycle — can help you understand these seemingly...Image re-used from Dong & Kantor, 2021 under CC-BY license. Figure 2 gives a detailed view of the structure...Hutcheson for her assistance with lentiviral replication.  Resources and References More resources on...101: Types of Viruses Viral Vectors 101: Viral Applications Resources on addgene.org Addgene's Lentiviral...

...Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive to small-molecules...fluorophore ligand to a peptide tag (Figure 1c). Click chemistry (e.g., transcyclooctene–tetrazine)– Nonnatural...toolbox of bioorthogonal chemical reactions (i.e., click chemistry) to attach a fluorophore at the specific... from fluorescent proteins given the relative simplicity of the system and the availability of fluorescent...labels for fixed-cell imaging, not for live-cell applications. Thus, these fluorophores are often bulky and...

...In the split-luciferase system, intein-mediated splicing reconstitutes functional luciferase only when ... luciferase protein interact and enable intein splicing (5). Screening with yeast two hybrid systems While... can often be accounted for using appropriate replicates and controls (see Tips below), and by validating...two-hybrid screening Minimizing false positives: Run replicates of the experiment to reduce the likelihood of...interactions in mammalian cells based on protein splicing. Anal Chem. 2001; 73:2516-2521. Pubmed PMID: 11403293...

...their experimental needs, and some of the common applications in the lab. Why Green Fluorescent Protein?...Re-engineering GFP to increase its range of color and application Almost as soon as its sequence was elucidated...others) Neutral mutations    A multitude of applications Due to its size and ease of use, GFP and other...visualize when and where the gene is expressed. Click here to view Addgene’s collection of empty backbones... just scratched the surface of the potential applications for GFP. It has also been used to identify particular...

...possibilities for the clinical and therapeutic applications of iPSCs. The generation of human iPSCs (hiPSCs...where each vector contains a viral origin of DNA replication, OriP and the EBNA-1 sequence that encodes for...are necessary and sufficient for retention and replication of the vectors in human cells [8]. The EBNA-1...cocktail. pCXWB-EBNA-1 lacks oriP and is unable to replicate in human cells, thus expressing additional EBNA...., Recent technological updates and clinical applications of induced pluripotent stem cells. Korean J ...

...identify mDCs and pDCs. Used under Creative Commons license.    In the third plot, anti-CD14 and anti-HLA-DR...different populations. Used under Creative Commons license.    Pro tip! Subtypes of immune cells are often...cells in each gate. Used under Creative Commons license.    Contour plots  FACS data can also be presented...Jhunjhunwala et al., 2015. Used under Creative Commons license.  Histograms  For analysis of a single marker...a flow experiment. Used under Creative Commons license.    Of course, there are many more things to...

...while freeze thaw is generally not recommended, Holic et al. found that lentivirus produced at pH 6 are... freshly collected lentivirus for downstream applications. While this ensures that the highest titer virus...establishes the best conditions for specific applications, stick with them! Changes in such variables ... 4.4 (2009): 495-505. PubMed PMID: 19300443. 7. Holic, N., Seye, A.K., Majdoul, S., Martin, S., Merten...lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer...

...EthoVision. Consult the literature to find all the applications of optogenetics relevant to your field. Ultimately...Inhibition of neuronal activation is a little more complicated since basal neuronal activity in your brain region...steps and technical considerations are directly applicable to the rat [6]. Technical differences include...rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement." Neuron 72.5...sensitivity optical neural silencer: development and application to optogenetic control of non-human primate cortex...

...live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP." Journal of the American Chemical...aptamer soup by exploring the development and applications of fluorescent RNA aptamers in Addgene’s collection...et al., 2017) and levels of the second messenger cyclic di-AMP in Gram-positive bacteria (Kellenberger ...fluorescent rna aptamer spinach2 for imaging of replication and immune-mediated changes in intracellular ...

...flanking the editing site on either side of the amplicon. Based on the frequency of out-of-frame edits...deleted accordingly. Pro tip: make sure your PCR amplicon isn’t too large so that you can easily visualize...sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is a software...expect off target editing to occur. Luckily, in silico prediction analyses like CRISPRitz or CRISPOR can...CRISPRitz: rapid, high-throughput and variant-aware in silico off-target site identification for CRISPR genome...

...immunofluorescence? Generally speaking, IF is an application in which antibodies that have been joined to ...distribute antibodies validate them in a variety of applications, including IF (or IHC or ICC), and will share...will share their findings either through their publications or as a review on a company website. It is important...Resources on the Addgene blog Learn about research applications of antibodies in Introduction to Antibodies ... for Me? Read about another common antibody application in The Basics of Western Blotting Resources ...

...multimerization of the PCR primers can cause duplication of the primer sequence in the resulting plasmid...proximal to the target site, can identify these duplications (slightly larger bands sizes relative to the...percentage agarose gel (~3%). Note that primer duplication will evade detection in the initial RFLP analysis...because the introduced restriction site is also duplicated. Validating your mutagenized plasmids Once constructs...identified (and confirmed to be free of primer duplications), final validation is performed by sequencing...