...Recently I learned that Addgene’s pLKO.1 cloning protocol is viewed around 3,000 times each month. I ...on the Addgene website in December 2006, the pLKO.1-TRC cloning vector (deposited by David Root of the...would the thousands of other scientists using pLKO.1 variants learn about those updates? This is a problem...manuscript to get reviewed and edited prior to publication, and this will delay the dissemination of your...deposited by George Church’s lab). Addgene has also cataloged a variety of useful molecular cloning protocols...bacterial transformation. And more plasmid-related educational resources can be found in our Plasmids 101 blog...bring your phone into the lab? Maybe having a dedicated lab-use tablet? The software also makes it easy...

...focuses on the replication machinery of Kaposi’s sarcoma-associated herpesvirus.       References 1. Snapp, ...experiments or even negatively impact cells (see Figure 1). When behaving properly, FPs enable researchers ... disulfide bonds in the secretory pathway (Figure 1) [4,6–8]. Additionally, FPs that encode N-glycosylation...compartments undergo significant post-translational modifications including glycosylation, disulfide bond formation...are equally susceptible to these non-native modifications, which can all potentially affect function [...N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering the protein... non-optimized parental proteins. Our recent publication illustrated the quantitative increase in fluorescent...

...(lenti) Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher...will help you decide which is right for you. Table 1: Mammalian versus insect cells for viral production...so most labs opt to purchase adenoviruses from dedicated adenoviral production labs or companies. Production...something you may have to optimize for your specific application. With mammalian systems, it is standard to do...more cells, while AAV can be titrated following purification using qPCR or digital droplet PCR. Insect cells...to collect your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in...titer the virus, or go straight into your viral application, like protein production. Titration of baculovirus...

...tethered to bring them into close proximity (Figure 1). Formation of the complete FP results in a fluorescent...detected by fluorescence microscopy. Figure 1: How BiFC works. A split FP (e.g., yellow fluorescent... protein interactions in living cells. Nat Protoc 1:1278–1286. https://doi.org/10.1038/nprot.2006.201 ...PIPs, resulting in a fluorescent readout.   Applications for bimolecular fluorescence complementation...resulting from the assayed PPI. This high-throughput application is much more straightforward to carry out because...protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described... supraphysiological expression levels. BiFC quantification This is where BiFC outshines FRET — BiFC experiments...

...environments such as live cells and tissues (Figure 1). These flexible strategies give you the best of both...majority of in-cell labeling strategies have two parts: (1) a genetically encoded “tag” expressed as a fusion...Follow him on twitter @rhodamine110.   References 1. Liu, Zhe, Luke D. Lavis, and Eric Betzig. "Imaging...Subsequent advances in fluorescent proteins have replicated many of the properties once exclusive to small-molecules...phosphopantetheinyltransferase) – These enzymes catalyze the covalent attachment of a fluorophore ligand...labels for fixed-cell imaging, not for live-cell applications. Thus, these fluorophores are often bulky and...the Addgene Blog Learn About Other Protein Tags Catch Up on Your Fluorescent Protein Background Which ...

... fusions that retain their individual activities (1). Researchers also discovered the modular format of...systems could be combined effectively (3). In Figure 1 for instance, the DBD of the fusion protein shown ...ubiquitin system has been devised (8). References 1. Casadaban M.J., Martinez-Arias A., Shapira S.K., ... 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...expression and correct folding, post-translational modification, protein degradation, access to the nucleus ...co-expression of the enzyme responsible for the modification in the assay host strain can help. In order...

...a rate of up to 1 per 500-1000 bp. In comparison, the DNA replication error rate is 1 per 109 bp. Unlike...variety of biological applications. First, GMAP-compatible backbones for lentivirus LV 1-5 and retrovirus ...Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved drugs against the entire kit; ...generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction...is multiplex automated genome engineering (MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate...of GFP (SEP) by a 9 amino-acid linker (see Figure 1). The key to the biosensor lies in pH: DsRed is relatively...inactivate SEP and leave only DsRed to fluoresce (1). With variants that can be targeted to specific cellular...

... Posts from Addgene 1) Twitter – Get your voice out there Here’s one thing you can do today: Sign up for...science communication are as varied as the field itself. Among other things, science communication comprises...certain aspects of art and education. If you suspect that science communication might be for you, don’t ... science education Do you aim to reach young people? Try networking with your local education community... Training – Get a formal education Some people get into science communication by taking a media or journalism...project. Science communication opens doors Getting involved in science communication has been the most...career researcher looking to break into science communication? Everybody goes about this in their own way....

.... Follow David on Twitter @driglar.   References: 1. Alberts, Bruce, et al. "Rescuing US biomedical research...Riglar Advocating for Science Symposium and Workshop 2016 – Sept 16-17 MIT, Boston The Advocating for Science...futureofresearch.org/advocating-for-science-boston-2016/. Sign Up For the Advocating for Science Symposium...Director of Media Communications, Harvard University Ray Howell -Founder of Howell Communications Philip Brenner...union dues. Time will tell the extent of the implications of this ruling and whether graduate students...labor laws, rather than as trainees in a special category, as has been the ‘labor-of-love’ philosophy of...tight lab budgets and even simply being able to categorize all postdocs at institutions, will be challenging...

...GpNLuc and OgNLuc are available through Addgene (Cat.# 70185 and 70186, respectively). Antares Chu, J...construct for Antares is available through Addgene (Cat. # 74279). Enhanced Nano-Lanterns  Suzuki, K., et...furimazine by Promega’s Chemistry group is a good story (1). From a deep sea shrimp to a compact powerhouse of...cellular analysis products at Promega.     References 1. Hall, M.P., et al. (2012) Engineered luciferase reporter...et al (2). In this post, I’ll cover two great applications of the NLuc technology. The first couples NLuc...fluorescence. The paper reports a wide variety of applications of the Enhanced Nano-Lanterns including monitoring...2017) Genetically encoded bioluminescent voltage indicator for multi-purpose use in wide range of bioimaging...

...This gives each guide a score ranging between 0 and 1. The cutting frequency determination (CFD score) is...converted to a weight (which ranges between 0 and 1) so that they can be used in the rank equation above...converted to a weight (which ranges between 0 and 1) (shown in the graph below) so that it can be used...HDR. Optimal mutation in-depth One the most sophisticated features of CrispyCrunch is how it mutates guide...

...microscopic resolution for studying colloidal gold (1).  The method was based on using a thin plane (sheet...Siedentopf, and R,. Zsigmondy.  (1902) Ann. Phys.  315, 1 – 39.
 A. H. Voie, D. H. Burns, F. A. Spelman. (1993...microscopy/en_de/products/imaging-systems/lightsheet-z-1.html ...of LSFM microscopes (3,4). After this initial application, Stelzer’s group described the single-plane or...brightness with increasing depth caused by light scattering and absorption.  Finally, the photomultipliers...

...the predecessor GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging...several other constructs in plasmid form. Fig. 1: Summary of the approach and outcomes of the screen...what you'll find in this post: Improved voltage indicator: JEDI-2P Novel class of light-gated potassium...-Myc [9E10] now available! Improved voltage indicator: JEDI-2P  by: Brian O'Neill  The François St-Pierre...have successfully developed an improved voltage indicator (GEVI) called JEDI-2P. Using a custom, high-throughput...screen for improved genetically-encoded voltage indicators (GEVIs) based on the ASAP sensors. The resultant...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408...

.... In literary terms, conflict can be defined as (1) the struggle between two opposing forces or (2) the...journey even when they know the ending.    Fig. 1: We all know that the superheroes win in the end. ...formal scientific communications, they are fantastic fodder for science communication. The trick is to ... is far from the only way to communication science. Science communicators also use the hero’s journey,...this small glimpse into the world of science communication and can use what you've learned to share your...Finding Conflict in Science  In all science communication, what you are doing is telling a (nonfiction...details often aren’t included in scientific communications, but they’re firmly part of the story to the...

... Figure 1: SPARC and SPARC2 use the GAL4-UAS system along with precisely truncated attP sites to... several common genetic tools in Drosophila (Fig. 1):  GAL4-UAS, a transcription factor binding site....of PhiC31. In the SPARC system, attP sites are located on either side of a stop cassette, upstream of ...amount of labeling, you can alter the attP site. Truncating the attP site upstream of the stop cassette results...population is directly correlated to the length of the truncated attP site. The three SPARC variants, based on ...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...Image from Isaacman-Beck et al., 2019. Applications for SPARC SPARC technology is versatile and can be ...

...into E. coli is a fairly inefficient process– just 1 out of 10,000 cells on average! Without some means...down especially fast and plates should be used witin 1 month for optimal efficiency. Beware of satellite ... perspective – a plasmid-containing cell must replicate the plasmid in addition to its own chromosomal...fully synthetic compounds. Antibiotics can be categorized based on whether they directly kill bacteria ...bacteriostatic); however, the distinction between the two categories may be a bit of a gray area as some bacteriostatic...Blocks 50S ribosomal subunit; inhibits aminoacyl translocation Bacteriostatic 50-100 ug/mL in EtOH Polymyxin...Binds 50S ribosomal subunit; inhibits peptidyl translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes...

...and take a look at each of its parts. Figure 1: Parts of an AAV transfer plasmid. Parts shown in ..., ubiquitous expression, while the human synapsin 1 promoter is neuronal specific (Haery et al., 2019)...contains the proteins needed for the virus to replicate,  and the transfer plasmid which contains the ... by the AAV. On a plasmid map, its sequence is located between the ITRs. It can be anything you like as...expression of your transgene to a specific species or location. Promoters can also drive different levels of ...sequences that can flank the transgene and are located between the ITRs. Transfer plasmids often have ... the expression of your transgene. The WPRE is located at the end of the AAV genome, just upstream of ...

... similar antibodies in llamas and alpacas (Figure 1). At the time, researchers were already on a quest...nanobodies (Arbabi-Ghahroudi, 2017)!      Figure 1: Comparison of a heavy-chain only antibody and single-domain...composed of camels, llamas, and alpacas (and their undomesticated counterparts) - not exactly species that jump...to sdAbs becoming valuable tools for diverse applications. For example, RANbodies can be used as an alternative... excitement around using sdAbs in diagnostic applications. For example, fluorescently and radioactively...heavy-chain only antibodies. (A) Overview of genetic modifications made to generate the nanomouse. (VHH = variable...inspired this versatile family of tools, the applications they have already enabled, and the discoveries...

...more tools for plasmid mapping and design (Figure 1). Benchling: While you might think of Benchling as...Davis from the University of Utah.   Figure 1: A plasmid map created in SnapGene of Blue Flame plasmid...tackling these particular tasks, but they tend to be scattered about the internet. To help you keep your virtual...: Find sequences for plasmids assembled from publications and commercially available sources, as well ... as expression patterns, post-translational modifications and polymorphisms Codon optimization tool: To...making it ready to copy and paste into other applications Primer design tools Many of the above-listed... well as primer design for different cloning applications. Primer3 and Primer3Plus: For general primer...

...from distantly related organisms. In 2006, less than 1% of the genes deposited at Addgene were synthetic....synthetic biology is expanding rapidly.  References 1. Kunjapur, Aditya M., Philipp Pfingstag, and Neil ...published their results in a paper in Nature Communications announcing a new bioinformatics tool that can...at its sequence. MIT collaboration for gene classification This inquiry started when Neil Thompson, a ...have generated robust tools that have practical applications in monitoring the accidental or intentional ...from farther away in the tree of life." Nature communications 9.1 (2018): 4425. PubMed PMID: 30356044. PubMed...the lab-of-origin of engineered DNA." Nature communications 9.1 (2018): 3135. PubMed PMID: 30087331. PubMed...