...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 ...

...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 ...

...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...

...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...

...approach may also be applicable to a number of rare genetic diseases caused by splicing defects, including...estimated that exon skipping therapies would be applicable to 80% of DMD patients. Figure 1: Exon...from Osterout et al. (2015b) under a CC-BY 4.0 license. Long et al., Nelson et al., and Tabebordbar...Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases. Nucleic Acid Ther. ...

... for PhDs or postdocs. Having this information publicly available may realign trainee expectations with...Publishing Panel brought in editors from a variety of publications as well as young PIs and all gave advice on ...blogging, write reviews, or write for a university publication, improving your general writing skills will help...cognizant of this during my PhD. Submitting a publication doesn’t have to be like throwing it into a black...Science The Future of Research Website Additional Publications of Interest Can Be Found Here Resources at ...

... as aliquots. Other serotypes may be available, click on the links for more information. Plasmids are ...

...adapted from Andrews et al., 2019 under CC-BY 4.0 license.   Selecting your antibodies When selecting...primary antibodies that have been validated in IF applications (find IHC validated or ICC validated antibodies...978-1-4939-8935-5_26 Jambor, H., Antonietti, A., Alicea, B., Audisio, T. L., Auer, S., Bhardwaj, V., Burgess...informative image-based figures for scientific publications. PLOS Biology, 19(3), e3001161. https://doi....Immunocytochemistry Protocols for other antibody applications...

...within that article you’ll find new plasmids. Simply click on the badge to be taken directly to the plasmids...account on PubMed. Once you have an account, simply click on your account name in the top right corner of ...PubMed to navigate to your account settings. Next, click on the link in the “NCBI Site Preferences” box. ...Preferences page, navigate to “PubMed Preferences” and click on the “Filters & Icons” link (see image to the ...

... wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human... Cas9 expressing lentivirus at the indicated multiplicity of infection (MOI). Anti-vinculin Western blotting...need to use FACS (like they did in the original publication). I thought I could see GFP expression with a...expression, but I didn’t. 2. In the original publication they see GFP expression in about 20% of their...an MOI of 0.3, as recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to target different...

...immunoprecipitation applications, but none of the traditional tags were suitable for both applications. To prove...These tags are easy to use in multiple common applications. Due to their limited effects on native protein...particularly in single-molecule or high-throughput applications. Perhaps IT5 and IT6 will even replace our older...

...Since the first research applications of GFP were published in the 1990s, biologists have spent a lot... Sattarzadeh et al., Sci Rep. 2015 CC BY 4.0 Applications and limitations of GFP photoconversion One concern...2):131-3.  PubMed. Find plasmids from this publication at Addgene. Subach OM, Patterson GH, Ting LM...nmeth.1664. PubMed. Find plasmids from this publication at Addgene.   Addgene’s Fluorescent Protein...

...funding and volunteer opportunities, protocols, publications, and more to accelerate the pace of research...is based in Canada, much of the information is applicable to researchers worldwide. “COVID-19 has spread...advance COVID-19 research to hopefully help guide policies and ultimately treatments in a timely fashion.... a significant impact on both clinical care and public health for this current COVID-19 pandemic, and ...

... QC/QA team as a DNA Sequencing Intern. The application process When I first saw the comprehensive job...immediately clear that Addgene wanted to ensure applicants understood what the expectations were for the...the intended position. After submitting my application, I was contacted to participate in a brief fifteen...the depositing labs intended. With the use of applications like SnapGene and Geneious, I was able to determine...