...discussed them in meetings and 2) creating a pictorial guide for our engineers (Fig. 2). Creating this guide was...in improving cross team communication has helped Addgene move from completing 2-3 large projects a year.... I needed to a) figure out the sources of miscommunication and b) find practical solutions.  My PhD ... for this task. Good scientists learn how to communicate their work differently based on their audience... family is going to vary widely. The way you communicate your science at a poster is going to vary depending...two main problems with Addgene’s cross team communication. Although these real world examples did not ... for the container to be used in cross-team communication A list of “AKA” names, or jargon terms, for ...

...gene-specific CRISPR and donor vectors as well as pX330S-2-PITCh, expressing a generic gRNA targeting the PITCh...Protocols, 2016 . Yamamoto Tagging Plasmids: pX330S-2-PITCh - expresses Cas9 & PITCh-gRNA pX330A-FBL/PITCh...RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled... supplemental materials from the Doyon Lab’s publication: Dalvai et al. Cell Rep. 2015 . Alternatively...programming sgRNA and HR donors for selectable genome modification. This system can be used to create C- and N-...following articles provide PCR templates for amplification of the tag (eg GFP, Flag, YFP, etc) and selection...

...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...DE3. For Phagocata morgani and Phagocata gracillis, the process is a little more complicated. These strains...Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracillis. There are 11 different ... coli. Thus, to get the dsRNA into Phagocata morgani and Phagocata gracillis, one has to purify the dsRNA... in situ hybridization experiments. A recent publication [1] from the Sánchez lab describes the plasmids...

...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid ...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage... incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...PMID: 38570691. Antibodies to facilitate SARS-CoV-2 research   By Ashley Waldron Ready-to-use recombinant...

...pLVX-EF1alpha-SARS-CoV-2-N-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 N protein  pLVX-EF1alpha-SARS-CoV-2-M-2xStrep-IRES-Puro...SARS-CoV-2 M protein pLVX-EF1alpha-SARS-CoV-2-orf3a-2xStrep-IRES-Puro: Expression of the SARS-CoV-2 ORF3a... SARS-CoV-2 protein expression The Krogan lab quickly shared plasmids expressing SARS-CoV-2 proteins at...pLVX-EF1alpha-SARS-CoV-2-E-2xStrep-IRES-Puro) and NSP2 (pLVX-EF1alpha-SARS-CoV-2-nsp2-2xStrep-IRES-Puro...months: Lentiviral preps for expressing SARS-CoV-2 proteins ChR2 for targeted expression DREADDs GFP ...Serotype Name 83899 AAVrg pAAV-mDlx-GCaMP6f-Fishell-2  Additional resources on the Addgene blog Read...availability date or to be notified when a particular catalog item is available for order please email [email protected]...

... to isothermal amplification by primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA...case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was able to detect as low ... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...easy-to-deploy, protocol for Cas13-based detection of SARS-CoV-2 genetic material (CREST) Sensitivity: 10 copies of...

....9 (parental) 46 +/- 0.7% AAV.PHP.S 82 +/- 2% Table 2: Transduction efficiency of AAV.9 versus AAV.PHP.S...figure 2) to allow for efficient IV delivery of virus to the CNS or PNS (figure32, tables 1 & 2). These...because: 1) their genomes are easy to manipulate, 2) they have long-term expression; and 3) they have ... On average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains ...stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction...cells are labeled with their own unique color; and 2) low levels of labeling so that a cell’s projections...maximize the number of cells that are labeled. Part 2: An AAV expressing the inducer (i.e. tet-off transactivator...

...permits nonspecific cation influx into the cell when stimulated. Channelrhodopsin-2 (ChR2), the first widely...domain2 of cryptochrome 2 (Cry2) from Arabidopsis thaliana is fused to truncated forms of cytosolic STIM1... (Phy) interaction 660 Cryptochromes Cryptochrome 2 and CIB1 Blue-light–mediated induction of protein ...interactions based on Arabidopsis thaliana cryptochrome 2 and CIB1 450 LARIAT Inhibits protein function by reversibly...customizable TALE DNA-binding domain with cryptochrome 2 protein and its interacting partner CIB1 to control... with single-spike temporal resolution. PLoS One. 2(3):e299. PMID 17375185 Kim B, Lin MZ. 2013. Optobiology...comparative analysis of microbial opsins. Nat Methods. 9(2):159-72. PMID 22179551 Saunders A, Johnson CA, Sabatini...

...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...can be seen exiting through the tissue. Therefore, 2-photon allows for deeper imaging into thick tissues...imaging objective that collects fluorescence. Like 2-photon microscopy, only a single focal plane of the...relying on raster scanning as is done in confocal or 2-photon microscopes. Total Internal Reflection Microscopy...

...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ...intended for the high level expression and purification of SARS-CoV-2 proteins for downstream experiments and...and codon optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV...Bioworks to synthesize and share over 1,850 SARS-CoV-2 plasmids to help academic and industrial labs accelerate...request? Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a...first deposited set of plasmids containing SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...

...Zhang GeCKO pooled library amplification 1 vector system: lentiCRISPR v2 2 vector system: lentiCas9-Blast...Mammalian Cell Lines CasPEDIA : An encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...computational pipeline that enables accurate quantification and visualization of CRISPR-Cas9 outcomes, ...off-target sites. This algorithm allows for the quantification of both non-homologous end joining (NHEJ) and...number of potential off-targets, target's genomic location, and genome annotation. Available genomes are ...sequence, which can be an accession number, genomic location, pasted nucleotide sequence, or a sequence text...

...Cre-ERT2 CAG Mammalian Heller 112622 pVHC_PGKneoLox2DTA.2 Venus, Cre-ERT2, targeting vector with MCS for homology...homology arms Mammalian Heller 112623 pEHC_PGKneoLox2DTA.2 Emerald, Cre-ERT2, targeting vector with MCS for homology... arms Mammalian Heller 112624 pmTHC_PGKneoLox2DTA.2 TFP, Cre-ERT2, targeting vector with MCS for homology...arms Mammalian Heller 112625 ptdTHC_PGKneoLox2DTA.2 tdTomato, Cre-ERT2, targeting vector with MCS for ...25;1504(4):467-86. doi:10.1016/0022-2836(81)90375-2. PubMed . Ventura A, Meissner A, Jaenisch R, Jacks... maintained). Translocation: If the sites are on separate DNA molecules, a translocation event is generated...recognizes 34 bp loxP sites, and the orientation and location of the loxP sites determines how the genetic material...

...comprehensive SARS-CoV-2 virus-human interaction network in which we expressed 26 of the 29 SARS-CoV-2 viral proteins...SARS-CoV-2 viral proteins except for Nsp3 and Nsp16, and includes an additional catalytically dead version...all became acutely aware of the threat of SARS-CoV-2. Like many in the science community, we’ve been motivated...identifying these potential human helpers of SARS-CoV-2 (especially the ones that are druggable) is a key ...just at the beginning of understanding how SARS-CoV-2 interacts with human proteins. For example, we used... joining in this worldwide fight against SARS-CoV-2. As we’ve all been affected in various ways by the...is going on there!).  Find the Krogan Lab SARS-CoV-2 plasmids (Note: The Nsp6 construct is undergoing QC...

...a slash, so that AAV2/5 indicates a virus containing the genome of serotype 2 packaged in the capsid from...efficiently transduced by AAV2/2, and is distributed more widely in the brain, indicating improved transduction... pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism...PJ, Mandel RJ, Muzyczka N. Mol. Ther. 2004 Aug;10(2):302-17. PubMed . From virus evolution to vector revolution...therapeutic applications. Clinical trials using AAV for various gene delivery applications are currently...adenovirus. These genes (E4, E2a and VA) mediate AAV replication. The transfer plasmid, Rep/Cap, and the helper...integrations may occur at a much lower frequency. As a replication-incompetent virus, AAV cannot enter the lytic...

...Cas enzymes? CasPEDIA is an encyclopedia of Class 2 CRISPR systems with wiki entries describing enzyme...Marker PI Publication Bacteria ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Plant ID...Marker PI Publication Yeast ID Plasmid Gene/Insert Promoter Selectable Marker PI Publication Zebrafish...RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled...editors are created by fusing Cas9 nickase or catalytically inactive “dead” Cas9 (dCas9) to a cytidine deaminase...and are thus well suited to directed evolution applications. Examples of these base editing systems include...Plasmid Gene/Insert Promoter Selectable Marker PI Publication Do you have suggestions for other plasmids that...

... non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several...Biol, 2003. 163(2): p. 257-69. PubMed PMID: 14581454. PubMed Central PMCID: PMC2173526. 2. Zacharias, D.... nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]....form dimers in a cell. The Kd of EGFP is 0.11 mM [2]. Following the simple logic outlined above, you might...organized smooth endoplasmic reticulum (OSER) whorls. 2)A dimerizing protein of interest fused to a dimerizing... designed to detect resulting in false positives [2, 4]. Solutions to the polymer problem There are some...Citrine) can be monomerized with an A206K mutation [2]. Addgene has many of these constructs and they are...

...AAV1, AAV5, AAV8, or AAV Retrograde serotypes and 2 x 10 12 GC/mL for the AAV2 serotype, as titered by...without a guaranteed titer. Any titer expectations communicated by email will supersede the general guarantees...step of the QC process, viral genome sequence verification, may not yet have been completed at the time...longer turnaround time. Any timing expectations communicated by email will supersede the general estimated...

...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just... phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. ...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways... studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed...members of their pathways, but where the kinases are located, their signal strength, and how long their signals...

...increase in virus. Purification (2 days) - Purification is required if rAdV is to be used in vivo. The standard...incubation as it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more... this “cytopathic effect.” Amplification can be repeated multiple times (2-4 rounds) at increasing scale...scale over the course of 1-2 weeks. Each round of amplification should result in a 10-100-fold increase...creating adenoviral vector constructs. It consists of 2 plasmids: a shuttle vector (in which the transgene...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...sequenced to confirm its integrity. Initial production (2-3 weeks) - Here you’ll produce the primary recombinant...

...the resulting fraction is reported. Figure 2: Normalization with loading controls. Under each lane...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears about the same...) are smaller than the analogous green “condition 2” points by the exact same value. For the two solid...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...factors we’ve covered in our previous blog posts (1, 2) on western blotting. If the abundance of your POI...