We narrowed to 401 results for: ARA-2

...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system." Cell (2015). PubMed PMID: 26422227... Find plasmids from this publication at Addgene. 2. Kleinstiver, Benjamin P., et al. "Genome-wide specificities...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell. 2015 Sep 23. pii: S0092-8674...also called Cas12a. In 2015, Feng Zhang’s lab characterized two Cpf1 nucleases, distant cousins of well-...Until recently, two major elements of Cpf1 characterization were missing: on-target and off-target editing...efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency of two Cpf1 orthologs...lower than that of the widely used SpCas9 and comparable to SaCas9. As seen with Cas9 orthologs, Cpf1 ...

... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...various papers to make the decision easier. 1. Müller, Sara M., et al. "Quantification of Förster resonance ...donor and acceptor fluorophores are fused to two separate proteins. (In contrast, intramolecular FRET probes...error in suboptimal or undetectable FRET, well-characterized FRET reference standards can be used to validate...

...procedure to be performed using a simple two-step, 2 hr protocol prior to transformation (Figure 1). As...Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid modification... for each type of modification is shown in Figure 2, and is discussed in more detail below. The main requirement...Biotechniques 10.1 (1991): 62-66. PubMed PMID: 2003926. 2. Bubeck, Peter, Monica Winkler, and Wilfried Bautsch...another simply amplify the vector and insert with separate primer pairs in the same PCR. Add homologous sequences...

...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an... the whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next...occurring animal model, the RPE65−/− dog, was characterized and found to suffer from early and severe visual...

... However, if you have a larger knock-in (200 bp – 2 kb), a dsDNA donor will need to be used due to synthesis...accidentally change your protein of interest.   Fig. 2: Stop re-cutting with PAM and guide RNA disrupting... the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR - Non-homologous.... Com., 8(2024), (2017). 10.1038/s41467-017-01836-2 Charpentier, M., Khedher, A. H. Y., Menerot, S., Brion...recognition sequence in the donor DNA, you can guarantee there won’t be re-targeting after HDR. If your...

...indicated they wanted to see more negative data (Figure 2.) At the same time, when scientists who support open...is not only housed, but encouraged.    Fig. 2: Results from a survey conducted by Addgene in 2021...detailed methods and image descriptions, a minimum n = 2, and well labeled high- and low-magnification images...gene therapy trials. Human Gene Therapy Methods, 26(2), 71–76. https://doi.org/10.1089/hgtb.2015.040 Initiative... in Adeno-Associated Virus Serotype 9 (AAV9) Preparations. Human Gene Therapy Methods, 30(6), 214–225....

...create the 2nd-generation lentiviral system (Figure 2). The HIV genes that do remain are very important ...1996 Apr 12;272(5259):263-7. PubMed PMID: 8602510. 2. A third-generation lentivirus vector with a conditional...CH, Humeau LM, Rebello T. J Gene Med. 2013 Feb;15(2):78-82. PubMed PMID: 23322669. 6. Genetic treatment... Banasik MB, McCray PB Jr. Gene Ther. 2010 Feb;17(2):150-7. PubMed PMID: 19847206. Additional Resources...Mulder GJ, Polack A, Bank A, Soulier J, Larghero J, Kabbara N, Dalle B, Gourmel B, Socie G, Chrétien S, Cartier...

... to DFHBI in the presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more...PMID: 25713381. PubMed Central PMCID: PMC4364232. 2. Baker, Monya. “RNA imaging in situ”. Nature Methods...emerging environmental pollutants.” Front Chem. 2014; 2: 41. PubMed PMID: 25019067. PubMed Central PMCID: ...biosensors.” TrAC Trends in Analytical Chemistry 27(2):108-117 · February 2008. 11. Svensen, Nina and Jaffrey...would bring the relevant ends together rather than separate them (Fig. 1b). One can also make “signal-off”...

...droplet in each well (Figure 2). A clean ddPCR should have a clear separation between positive (blue) and... to 2 minutes and the denaturation time to 1 minute has been shown to increase droplet separation (Witte... qPCR is that the results can vary by a factor of 2. This means that if you set up two identical assays...getting a clean separation between positive and negative droplets with these parameters, here are a few...Since Addgene began producing AAV vector preparations, we have used quantitative PCR to evaluate the ... for ddPCR set-up. Prepare the master mix in a separate area from where you prepare your sample dilutions...your PCR For a good starting place on what PCR parameters to use, see Lock’s seminal paper on AAV titration...

...3 plates at different densities for selection). A 2% agarose solution is then melted (and thereby sterilized...96-well assay plate. We generally freeze at least 2 ampules each of 4 clones with different levels of ...of agarose, which is then overlaid with the firmer 2% agarose gel solution discussed above for picking....concentrations of salts and glucose. If we circled well-separated clones containing more than 50 cells (visible ... should contain at least 50 cells and be well-separated. If the growth rate/transfection efficiency is... with this!) Make sure your colonies are well separated. For this, you need to have some idea of the rate...individual cell clones with different expression characteristics from a population of transfected clones. The...

...preliminary risk reduction at a minimal cost. Step 2: Rough screening of nuclease variants by next-generation...different ranges of the activity spectrum (Figure 2). Identify the least active variant that still shows...test if you want further refinement.  Figure 2: Schematic representation of the two-step screening... last active and the first inactive one from step 2. (In the example in Figure 3, the least active still...applications where the highest achievable specificity is paramount: Pick and test some more variants from the CRISPRecise...Tusnády, G. E., & Welker, E. (2021). A method for characterizing Cas9 variants via a one-million target sequence...

...phosphorylation events in the proteomes of higher mammals (2). How can we begin to understand the importance of... the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone of the phosphoserine...optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial host strain for pSer-incorporation...biology4.5 (2002): E127-E130. PubMed PMID: 11988757. 2. Hornbeck, Peter V., et al. "PhosphoSitePlus, 2014...phosphorylated intermediate of a protein for characterization. However, phosphoproteins are often low abundance...strategies; expression should be optimized for parameters such as induction temperature and length of induction...

...mammal called alpaca could neutralize the SARS-CoV-2 virus. But while the early results were promising,...receiving dozens of requests every day.    Figure 2: When a researcher needs a collaborator for a specific...Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the expert’s suitability and availability...diseases, so we were left with no choice but to quarantine in our houses, feeling helpless about the situation...

...to (1) run a DNA gel to confirm fragment size and (2)  incubate with RNAse and Proteinase K to purify the...proteins of interest to any DNA they were bound to (2) fragmented the DNA (3) used antibodies to specifically...adjustments, to get everything just right!  Fig. 2: Goldilocks is actually celebrating a successful ChIP...remain bound to each other until you are ready to separate them. Most proteins are not likely to remain attached...

...the result in Figure 1 could look like this: Figure 2: Expression of Plasmid B (as internal control) This...down expression of Gene X but, as shown in Figure 2, this was likely due to the original transfection ...PMID: 20074371. PubMed Central PMCID: PMC2817684. 2. Replicates and repeats—what is the difference and...multiple separate wells (within the same plate) with purified plasmid from the same aliquot/preparation. The...co-transfected with the test plasmid or transfected into a separate well of your cells. Regardless, the amount of ...should be consistent over time and from different preparations of the purified test plasmid expressing the ...Using our example above, we would test different preparations of Plasmid A in different aliquots of cells ...

... Jan;20(1):81-89. PubMed. 2. Bauer DE, Kamran SC, Lessard S, Xu J, Fujiwara Y, Lin C, et al. An erythroid...with TALENs, in collaboration with the Porteus lab [2]. With the advent of CRISPR/Cas9, we began to explore... inferred based on correlative biochemical characteristics and ectopic reporter assays. We have been interested..., identifying these deletions, and finally characterization. Read and download the full protocol from ...Dass A, Yien YY, Chung J, Masuda T, et al. Characterization of genomic deletion efficiency mediated by...

... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis... to be explored and exploited. With better characterization, these proteins could function as surrogate...surrogate antibodies for identifying and characterizing sugars and sugar-containing molecules in molecular biology...colonies of interest, sequence the inserted DNA and characterize your new putative host cell-binding proteins...

.../10.1038/s41587-024-02268-2 Schep, R., Brinkman, E. K., Leemans, C., Vergara, X., van der Weide, R. H.... flanking sequences to their input string (Figure 2). This string can then be used as input for the prediction...top 5) and test them in their labs. Figure 2: Guidelines to predict pegRNA efficiencies with pridict.it...we can evaluate tens of thousands of designs in parallel. To do this, we performed high-throughput ‘self-targeting...the TRIP (Thousands of Reporters Integrated in Parallel; Akhtar et al., 2013; Schep et al., 2021) method...Assayed by Thousands of Reporters Integrated in Parallel. Cell, 154(4), 914–927. https://doi.org/10.1016...., Benvenuto, E., Schmidheini, L., Schep, R., Damodharan, T., Balázs, Z., Janjuha, S., Ioannidi, E. I....

...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...vectors to answer this question, mapping and characterizing the activity of the DA neurons of the VTA. ...brain area, demonstrating that it is possible to separate neuronal inputs to induce aversion- or reward-...PMC4971574. Hooper, Andrew, and Jamie Maguire. "Characterization of a novel subtype of hippocampal interneurons...

... delivered separately). Figure 5: Mechanism of ATLASCre. VAMP2 = synaptobrevin 2; At = ALFA-...RNA guide (known as an omega RNA, or ωRNA) (Figure 2), but TnpB is small enough to be carried by viral ...than a third of the size of Cas9). Figure 2: Comparison of TnpB (A) and Cas9 (B) genome editors...mGold2t (Lee & Lai, et al., 2025). The team characterized and deposited a variety of constructs for visualizing...TnpB (named ISYmu1) to the popular plant model Arabidopsis (Weiss et al., 2025). By co-delivering TRV1 with...editor for transgene-free germline editing in Arabidopsis. Nature Plants, 11(5), 967–976. https://doi.org...

...its abdomen to produce yellow-green light (Figure 2). Despite the title of this post, this reaction is...; no chemical reaction takes place. Figure 2: Simplified chemical reaction catalyzed by luciferase... has a detailed list of luciferase options. Table 2: Properties of luciferase enzymes Name Natural ...interaction occurs, the luciferase emits its characteristic light. However, if a protein interaction brings...

...promoter and terminator flanking the CDS, and a Level 2 multigene vector with two other independently regulated...the sequence you are trying to clone. Figure 2: MoClo assemblies depend on compatibility of four-...sequentially for your Level 0, Level 1, and Level 2 assemblies using the plasmid sequence files provided...after each transformation. This could be as few as 1–2 colonies if you’re an experienced MoClo user, but ...MoClo kit through their online catalog pages. We guarantee this will save you a lot of headaches down the...

...) 38 kb Cells HEK-293T Sf9 Time to collection 2–4 days 5–10 days Purification Ultracentrifugation...Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher Lower Resources... a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https...transfection). This differs from typical insect cell preparations, which we talk about below. Processing The viral...Plasmids and Resources References Shaw, G., Morse, S., Ararat, M., & Graham, F. L. (2002). Preferential transformation...

...experiment using smFPs and standard neuronal tracers. 2. Visualization of neuronal sub-cellular structures...similar proteins. For example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays...PMID: 25915120. PubMed Central PMCID: PMC4573404. 2. Shepherd, G.M. (2004). In "The synaptic organization...STORM imaging, and electron microscopy. Sample preparation for these techniques sometimes makes it more ...retain their ability to be detected after sample preparation better than standard proteins tags like GFP. ...smFPs in the future. References 1. Viswanathan, Sarada, et al. "High-performance probes for light and ...

...properly exiting the ER and fluorescing (see Figure 2) [5].  When using FPs containing two cysteine residues...compartments including the secretory pathway (Figure 2), the inner membrane space of mitochondria and chloroplasts...PMID: 18228466. Pubmed Central PMCID: PMC2875081. 2. Snapp, Erik Lee. "Fluorescent proteins: a cell biologist's...inherent brightness and spectral characteristics of the moxFPs are comparable to the non-optimized parental...protein activity. We encourage investigators to characterize FP-fusion proteins functionally relative to ...

...indicating primer dimers. (c) A double peak indicating 2 amplicons. Image credits (respectively): Zuzanna K...details for reproducibility’s sake!   Fig. 2: Setting up triplicates in multiple configurations...used method is the Standard Curve method. See Table 2, (adapted from Wong, 2018) for a comparison of different...Statistically defined log-linear phase   Table 2: A comparison of different qPCR analysis methods. ... for RNA quantitation. Biotechniques. 2000 Aug;29(2):332-7. doi: 10.2144/00292rv02. PMID: 10948434.Taylor...(Taylor, 2017). With the right conditions and parameters, some people find it can detect even smaller ...run a melt curve to validate your primers. This separate thermocycle allows you to measure the intensity...

...revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central... to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A....adapted from Zhou and Melton, 2008, [43]): Characteristic Reprogramming+ Transdifferentiation in vivo...

... environmental sustainability in mind (Table 2). Table 2: Sustainability features highlighted in plasmid... and then clean-up the plasmid DNA (Figure 2). For most kits, the steps for lysing cells and removing ...together — no mixing and matching!   Figure 2: Plasmid isolation workflows are similar across kits...Sold Separately Yes, buffers and columns sold separately Some, waste tubes sold separately No...carry-over into the eluate, while other kits are guaranteed to yield endotoxin-free DNA. Once you know the...stages: lyse the bacteria harboring your plasmid; separate plasmid DNA from unwanted cellular “debris”; and...kits. Lyse your bacteria to release the plasmid, separate the cell debris from the plasmid, and then clean-up...

...under phase contrast starting day 2 after transfection.     Fig: 2: Expressing mARGAna in MDA-MB-231...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the...for longer. Cultures induced with arabinose and colonies on arabinose-containing plates will be visibly... inducible expression systems in E. coli. The arabinose-inducible promoter pBAD and a low-copy number ...wide range of inducer concentrations (0.1-1% L-arabinose) without signs of toxicity or burden relative ...or on LB-agar plates containing glucose and L-arabinose at 37oC for at least 12 hours, or at lower temperatures...after the E. coli have colonized tumors, inject L-arabinose intraperitoneally and wait 24 hours before imaging...

...ITR, which can either be “flip” or “flop” (Figure 2). Additionally, the ITR contains a 4-nucleotide Rep...site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type ITR in flip and flop orientation. ...Packaging, and Potency of AAV Vectors. Microorganisms, 12(2), 310. https://doi.org/10.3390/microorganisms1202031038399714..., B-B’, and C-C’) that give the feature its characteristic T-shape. The arrangement of the B-B’ and C-...

...A-B+ cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive... the different populations. In the case of Figure 2, one population is B- and another two are B+. Our ...are in the lower right corner.      Figure 2: This flow plot has two populations that are B+ and...actual flow cytometry run, all markers are read in parallel.  Let’s imagine we’re looking for cells in a sample...markers to most specific, the first step is to separate cells from debris and roughly group the cells ...gated population, two types of FSC are used to separate single cells from doublets, ignoring anything ...population of B cells and dendritic cells is further separated into three different populations using conjugated...

...partitioning each type of plasmid into a separate daughter cells (Figure 2) (Ebersbach et al., 2005). However...to plasmid loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids...Plasmids. Microbiology and Molecular Biology Reviews 62(2): 434–464. Pubmed PMID: 9618448 Diaz R, Rech J, Bouet...model, termed the “handcuff” model, iterons of two separate plasmids bind to the same RepA molecules, forming...NTPase is recruited and “walks” each plasmid to a separate daughter cell. When plasmids are compatible, different...bind to each plasmid type, and different NTPases separate the plasmid pairs into the new daughter cells.... Pubmed PMID: 22153351 Shintani M, Sanchez ZK, Kimbara K (2015) Genomics of microbial plasmids: classification...

...evolutionary tree at least 6-7 times and on at least 2 separate continents. Mimulus (Andrea Sweigart) Monkeyflowers...presented in a series of quick (10 minute) or very quick (2 minute) talks so we got to hear from everyone. Addgene...Organisms, Part 1 Five Popular Model Organisms, Part 2 .... Sticklebacks demonstrate unusual parallel adaptation to disparate environments so can be used study ...difficult). Cuscuta (James Westwood) Dodders are parasitic plants that steal all their food from other plants...have economic importance as harmful weeds. The parasitic fusion is such that the two plants share RNA, ...

...discussed them in meetings and 2) creating a pictorial guide for our engineers (Fig. 2). Creating this guide was...communication has helped Addgene move from completing 2-3 large projects a year, on average, to completing...examples did not happen in the lab, they have parallels to collaborative lab work. Corralling context...

...CcdB.” Biotechniques. 1996 Aug;21(2):320-3. PubMed PMID: 8862819. 2. Bahassi, EM., et al. “F plasmid CcdB... desired insert should not grow.   Figure 2: Image from http://teachline.ls.huji.ac.il/72682/tutorials...advantages that will be discussed in detail in a separate post. Focusing on the ccdB aspect, Gateway takes...

...-induced changes in root-to-shoot ratio in Arabidopsis, (2) expression patterns in plants with enhanced... go back and finish the review.  Peer-review step 2: Detailed assessment After this initial assessment... of Arabidopsis HapMap population in Amsterdam. Currently, she is a postdoc at KAUST, Saudi Arabia, in...Julkowska, a postdoctoral researcher at KAUST, Saudi Arabia. From the perspective of an author submitting ... specific repositories (Addgene, WormBase, Arabidopsis.info). These repositories will ensure that the ...guest blogger Magdalena Julkowska from KAUST, Saudi Arabia. Magdalena Julkowska did her Ph.D. on salt-stress...

...resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after herbicide was ... CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression... when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET... to drive prime editing in both tomatoes and Arabidopsis, suggesting the improvements may be broadly applicable...enables heritable desired edits in tomato and Arabidopsis. Nature Plants, 10(10), 1502–1513. doi: https...

...improve solubility and stability (Figure 2).      Figure 2: Crystal structures of CRBNmidi alone...fluorescent tags in Caenorhabditis elegans. Genetics, 228(2), iyae126. https://doi.org/10.1093/genetics/iyae126...K., & Russell, W. C. (1987). Isolation and characterization of monoclonal antibodies to simian virus 5...matured considerably faster, with expression levels comparable to GFP during early embryogenesis (Figure 3)....Merritt D.M., Pe K., Cesar M., Hobert O. (2024) Comparative analysis of new mScarlet-based red fluorescent... and the LgBiT luciferase fragment (expressed separately) assemble and catalyze the luminescence reaction...

...host range plasmids. FEMS Microbiology Letters, 348(2), 87–96. https://doi.org/10.1111/1574-6968.12241  ...mobilization of broad host-range IncQ plasmids. Plasmid, 62(2), 57–70. https://doi.org/10.1016/j.plasmid.2009.05.001...negative hosts. Biotechnology and Bioengineering, 106(2), 326–332. https://doi.org/10.1002/bit.22695  Schmidhauser...Understanding the characteristics of natural plasmids and how they function in nature can inform us on...trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccous denitrificans, Pseudomonas spp., Rhizobium spp...Moraxella spp., Mycobacterium aurum, M. smegmatis, Paracoccus denitrificans, Pasteurella multocida, Porphyromonas...Escherichia coli, Gluconacetobacter xylinus, Paracoccous denitrificans, Pseudomonas fluorescens, P. putida...

...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...fluorescent proteins to find those with ideal characteristics (low aggregation, high photostability, and ...