...cancer. These viruses need to be handled in a BSL-2 (or BSL-2+) lab and require additional safety measures.... BSL-1. In BSL-2 labs, eye protection is required any time you have to work with BSL-2 materials, such...diseases in humans, earning this virus a BSL-2 classification. Lentivirus (a type of retrovirus) and gamma...recommended or required in BSL-1 and required in BSL-2 labs. When working with lentivirus, many organizations...sprayed into the air. For lentivirus and other BSL-2 materials, you’ll need to use aerosol-tight caps, ...percentage of the decontaminate to be used for BSL-2 waste, and of course, if you are working with a substance...host genome, the virus is replication incompetent, meaning it can’t replicate and continue the infection...

...safely. Recently, many SARS-CoV-2 researchers began pseudotyping the SARS-CoV-2 spike protein into a lower ...SARS-CoV-2 virus. Scientists have been using similar pseudotyping methods with other viruses in a BSL-2 lab... et a., 2018).   Figure 2: To pseudotype with the SARS-CoV-2 spike protein, scientists encode...production uses three plasmids: (1) The transfer plasmid, (2) the envelope plasmid, and (3) the packaging plasmid...example, production of vesicular stomatitis virus, HIV-2, and human foamy virus in cell lines expressing galactosyl...for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 12...Are Inactivated by Human Serum. Molecular Therapy 2:218–222 . https://doi.org/10.1006/mthe.2000.0116 Gutierrez-Guerrero...

... go back and finish the review.  Peer-review step 2: Detailed assessment After this initial assessment...salt-induced changes in root-to-shoot ratio in Arabidopsis, (2) expression patterns in plants with enhanced sodium... to help decide whether work is suitable for publication in their journal. The early peer-review attempts...information that would prevent the study to be replicated or omission from the literature. I think that...Addgene.org Learn about depositing plasmids pre-publication Find molecular biology protocols Find eBooks ...

...detect SARS-CoV-2, including many with plasmids deposited at Addgene! 2. DNA Purification Without a Kit...detection methods, synthetic nanobodies, SARS-CoV-2 protein expression, and more. We also highlighted ...empty backbones, fluorescent protein tags, SARS-CoV-2 plasmids, etc. It is our hope that by providing cloning...RNA and extracting RNA without a kit. 3. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas by ...virtually. 6. Quantifying DNA? Here are Five DNA Quantification Methods to Consider by Jennifer Tsang The...The nanodrop vs. spec debate dominates DNA quantification discussions, but what about the other methods...

...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required for recombineering...generic lambda red recombineering experiment (Figure 2). In the following sections, key steps that differ...confirmation of recombinant clones.    Figure 2: Overview of using Lambda Red recombineering system...pairs of homology to the targeted insert site (Figure 2, top).  These primers are typically ~70 nucleotides...bacteria that has key MMR proteins knocked out or 2) specially design ssDNA oligos to avoid MMR: 1) E....will have more unintended changes to their genomes. 2) Designing ssDNA oligos that avoid MMR activation:...

...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic...similar applications and have similar advantages, though with some caveats.  First off, F(ab’)2 fragments...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth.2022.100301...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems.... (2019). Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. ...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...

...Another stabilizing sugar modification, found at the same site, is 2’-fluoro (2’-F), which substitutes a...phosphorothioates with 2’-O-methyl modifications, proved more stability than just one modification alone (Hendel... with post translational modifications. One of the most common of these is 2’-O-methylation, where a methyl... the 2’ position. Moving one position over in the sugar ring, a popular stabilizing modification is 3’... methyl group is added to the 2’ hydroxyl group of the ribose to protect it from nucleolytic attack. Another...generated by introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting region of...switches. Stabilizing gRNA modifications  Sugar phosphate backbone modifications The recognition sequence...

... Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series that ranges...series ranging from 1:2 to 1:32. Each individual step of the series is consistent at 1:2. When preparing a.... An example of this is shown in Figure 2.   Figure 2: The concentration of a serially diluted... ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration of ...accuracy of the curve, avoid pipetting small volumes (< 2 µL) or volumes too large for a standard micropipette... be run in duplicate or triplicate. The closeness of the data points generated by replicate values provides... the standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve...

... and G-protein is delivered to the starter cell. (2) The starter cell now expresses TVA and G-protein....which is not required for the transcription or replication of the genome within infected cells, but is required... cells that lack the G protein, the virus can replicate but the newly formed viruses are unable to infect...starter cell via the EnvA receptor. RABV is able to replicate in the starter cell using the G-protein, which...offers you even more options. Select from a vast catalog of eligible AAV plasmids and we’ll make the viral...

... Human M3 muscarinic Arrestin-2/-3 CNO* Increase Arrestin translocation Arrestin signalling Nakajima &...Brown KM, Forbes SL, Liggett SB (2001). Modification of the beta 2-adrenergic receptor to engineer a receptor-effector...muscarinic receptor and has not been used in vivo . Figure 2: DREADDs, their ligands, and signaling properties....hM1Dq hM5Dq Human M3 muscarinic G αq CNO* Increase Ca 2+ Neuronal burst firing Armbruster et al., 2007 rM3D...channels, their effect, and outcome in neurons. Table 2. Descriptions of PSAMs and their activity in neurons...neurons) that can be turned on or off by the application of a small molecule ligand. The ideal chemogenetic...chemogenetics for research and potential clinical applications. Science. 364(6436):eaav5282. PMID: 30872534...

...Durocher et al. Nucleic Acids Research 2002 Jan 15;30(2):E9 A list of commonly-used bait and prey plasmids...benchmarked protein microarray-based platform for the identification of novel low-affinity extracellular protein...

...plasmid Bullock and Port 49409 pCFD2-dU6:2gRNA dU6:2 BbsI Injection or in vitro transcription Virmilion...ID Plasmid Gene/Insert Promoter PI Publication Activate Catalytically dead dCas9 fused to a transcriptional...RNA Targeting RNA Targeting RNA Editing Other Applications Purify Tag Visualize dCas9-FokI Screen Pooled...introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs ...than NHEJ. ID Plasmid Gene/Insert Promoter PI Publication Nick CRISPR/Cas nickase mutants introduce gRNA-targeted...repair (HDR). ID Plasmid Gene/Insert Promoter PI Publication Prime Edit Cas9 H840A nickase fused to a reverse...specific locus. ID Plasmid Gene/Insert Promoter PI Publication Empty gRNA Expression Vectors Select a gRNA expression...

...GEVIs (called ASAP 1 and 2), they found an indicator that is optimized for 2-photon imaging of voltages...   Fig. 2: A) HcKCR1 is highly selective for K+ over other Na+ and other cations. B) Cortical slice...resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations. Image...slice of HcKCR1-EYFP and tdTomato expressed layer 2/3 neurons in mouse. C) Action potential frequencies...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...

...Information Featured Collections COVID-19 SARS-COV-2, ACE2, and CRISPR tools Fluorescent Proteins from ...antibodies are produced in-house and undergo application-specific validation and quality control by Addgene... Recombinant Antibody Reactivity Recommended Applications PI Don’t See What You’re Looking For? Please...within 1 business day regarding your account verification. Once your account has been verified, you will.... Pricing Addgene is a nonprofit organization dedicated to providing the scientific community with open...access to plasmid research tools. Pricing will be indicated on the product pages after you login to your account... the depositing institutions. These agreements indicate the language which we are allowed to use for distribution...

...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Lock,...PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal... virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant and/or producer..., R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: Packaging of genomes... genomes can limit production of recombinant AAV-2. Gene Therapy, 6(7), Article 7. https://doi.org/10.1038...Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...

...against GFP expression, but I didn’t. 2. In the original publication they see GFP expression in about 20%...duplexes (513 bp), as indicated by the presence of a 428 bp digestion product (lane 2). The undigested (-...transductions and use a range of reasonable MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially...doing a screen. I made a few monoclonal lines (Figure 2) and I was surprised at how variable Cas9-expression...across the different cells (Figure 3).   Figure 2: Cas9 expression in monoclonal cell lines generated... expression Overview: By Western blotting (Figure 2, pool), I knew the cells expressed Cas9, but I didn... appear green despite having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this...

...260 and 280 nm should fall ~1.8 - 2 and give scientists an indication of purity versus protein contaminants...260 and 230 nm ratios should fall ~2 - 2.2, and provide an indication of purity versus chaotropic agents... be extracted from small bacterial cultures (i.e. 2-3 ml) with or without commercially available kits ...hydrochloride used during plasmid extraction. Figure 2: Use cloning grade DNA for direct cloning of your ...that have been used during the purification procedure. Good indicators of DNA purity are the absorbance...

...the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed to...and leave exon 2 floxed, as seen in line 4 of Figure 4. Since the loxP sites are located in intronic regions... through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting...For recombination to occur in a cell, a minimum of 2 kb of sequence homology is required, but 6 to 14 kb...construct to your target cells. After recombination, exon 2 of the targeted gene will be removed from the chromosome...the GFP/NeoR cassette is inserted in place of exon 2. Thus, the targeted gene is disrupted (knocked out...

...PMID: 29263082 Find plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., ...antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough...enough that multiple labels (green circles in figure 2 below) can be attached to each antibody. But being...displacement Require separate incubation of 1° and 2° Must use different species and/or different IgG ...Greater resolution due to lower label displacement 2° incubation can be skipped by pre-binding to 1° ...a fluorescent tag and the target antigen to about 2 nm, which can lead to higher resolution for super-...subclass.   Express the Nanobody In Bacteria: Table 2 also has a link to the plasmid you’ll need to express...