...from GFP and the mutation definitions found in Table 2 may not apply. Table 1: The specific mutations comprising..., V163A EBFP F64L, S65T, Y66H, Y145F   Table 2: Functional role of specific mutations in GFP derivatives...biochemical properties. In 1995, Roger Y. Tsien described an S65T point mutation that increased the fluorescence... in the wildtype protein and facilitating its widespread use in research. Many other mutations have since...array of GFP-based fluorescent biosensors has been designed to detect a variety of intracellular conditions...

...days before the deadline isn’t going to be much use. 2. Originality is essential for grant success The two... it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback from...to a lay audience. My lay paragraph typically includes a couple of sentences of introduction to my area...

... reduce the background staining (Table 2).      Table 2: Secondary antibody selections for a four-antibody... and to work in a variety of applications. The desired specificity of your secondary antibody depends ...

... of permeabilization and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol ... at too high a concentration.      Figure 2: Examples of different permeabilization strategies...improve antibody binding for some targets, they can destroy it for others. That said, organic solvents’ impact...critical to the outcome of your IF experiment and deserve careful thought and attention.   * There are other...

...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression...Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes is currently...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)... large size of Cas9 activators, which largely precludes delivery by adeno-associated vectors (AAV). This...packaging capacity than AAV. Such an approach was described recently by the Day Lab, who developed neuron-...

...connectivity to other data sources used in research; (2) connectivity to other tools used in research; and...by making it easy to get data out of the ELN; and (2) facilitate inter-operability with other research ...reproducibility than others. Unfortunately, most ELNs are designed as closed ecosystems and act as data silos because...RSpace ELN supports reproducibility RSpace was designed in response to requirements that emerged from ...used in research, and data repositories. Below I describe how each of these kinds of connectivity facilitates... in institutional and lab servers specifically designed to hold large amounts of data. Although it may... use of other tools they use in research. This includes chat apps like Slack, Microsoft Teams, and Google...

...important role in solving the structure of DNA. 2. The Immortal Rap of Henrietta Lacks Students in Oakland...One of Tom's earliest and most popular videos. Describes fossil rocks and their importance in understanding...

... which one you choose depends on your experiment (de Ruijter et al., 2003). For example, we did our initial...GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate, which will then...Humana Press, 2009. 41-50. PubMed PMID: 19183892. De Ruijter, N. C. A., et al. "Evaluation and comparison...and a vacuum is applied with a vacuum bell or desiccator. Successful infiltration can often be seen by...

...et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by QCascade; although...cells of the plasmid after integration. Figure 2: (A) Streamlined VchINT expression constructs. (B)...human therapeutics (e.g. using Sleeping Beauty). Despite their diverse utility, DNA transposons have been...(V. cholerae INTEGRATE - hereafter VchINT) and encodes a Type I-F CRISPR-Cas system. The four major components...the same promoter, and a donor plasmid (pDonor) encodes the mini-transposon. pEffector and pDonor can also...: Clone spacer(s) into the CRISPR array Clone desired DNA cargo into the mini-transposon Transform plasmid...even very large genetic cargo, at one or multiple desired genomic loci. Many applications of these systems...

...expressed as a fusion with your favorite protein and (2) a synthetic fluorophore-contianing “ligand” that ...cell 58.4 (2015): 644-659. PubMed PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve...individual molecules. Poor photon budgets are a widespread issue with fluorescent proteins—even the thriftiest...student searching couch cushions for spare change, desperate to extract a few more photons from a sample. Chemical... the complexity of the system. A particularly desirable property for small-molecule labeling strategies...size, brightness, and cell permeability. In past decades, further refinements to this established dye structure...spectral range. Nevertheless, these fluorophores were designed as antibody and oligonucleotide labels for fixed-cell...

...of these neurons via the channel Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC...deliver Cre-dependent expression of  Channelrhodopsin 2 (ChR2), a modified receptor protein that activates...Over the past decades, neuroscientists have made great strides to map brain regions and allocate specific...for this task. However, most lesioning methods destroy areas larger than intended and may unintentionally...surface. The latter reflects a state of behavioral despair, and the more time the animal spends in the passive...that were not (Hare et al., 2019). The research described here is only a small selection of the increasing...

...diffusion test or Kirby-Bauer susceptibility test (Figure 2), and requires at least 18-24 hours to obtain results...." London: Wellcome Trust & HM Government (2016). 2. CDC Information Pages on Antibiotics 3. WHO Antimicrobial... defined as microbial survival despite exposure to antibiotics designed to kill them or to impede their...that the term ‘antibiotic’ can also be used to describe anti-parasitic drugs and anti-fungal drugs, because...different reasons. Exposure to antibacterial drugs provides a selection pressure, killing off bacteria that...equipment, etc. Virtually any preventive measure designed to help keep you from getting sick can be helpful...

...Halloween is only 2 days away and here at Addgene we couldn’t be more excited! As a small, tight-knit...occurring earlier but that the photo evidence was destroyed to protect the innocent revelers. Our Halloween...for Halloween and how seriously we take costume design. In case you need more convincing, I’ve got just...to great customer support by dressing up as our Zendesk email system.  Our 2013 winners dressed up as ...

...antibiotic selection. Essentially, this gene is a 2-in-1 providing both bacterial and eukaryotic selection...Zeocin-induced DNA cleavage in human cells. Mutagenesis, 20(2), 111–114. https://doi.org/10.1093/mutage/gei016I ...The NPTII gene confers resistance to several aminoglycosides, including G418 which is commonly used for ...DNA and generating DNA breaks. The Sh ble gene provides zeocin resistance by expressing a protein that...experiments in which off-target mutations are undesirable. Pros: Works across many species/systems. Cons...

...your sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample that will link biomolecules...quite so much. Created with biorender.com Steps 2-4 are not found in standard IHC/ICC protocols and ...Microscopy. What is Expansion Microscopy?  ExM provides a straightforward answer to the space problem....improve label density (Mikhaylova, et al. 2015).  Despite these limitations, ExM is a valuable tool, especially...Bournonville L, et al (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria ...

...green squares, purple circles and blue triangles. 2) An antibody immobilized on a bead binds specifically...protein G, a novel IgG-binding reagent. J Immunol 133(2):969-974. Hjelm H, Hjelm K, Sjöquist J (1972) Protein...most situations.  A slightly different method is desirable if the antibody is expected to bind weakly to ...vary in shape and structure. Their porous surface provides a large area for binding, but antibodies conjugated... slightly higher, than those of agarose beads, despite the size difference. Magnetic beads are particularly...

...detection, and control. Int J Pharm. 2018 Oct 25;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub...and particle formation. J Pharm Sci. 2015 Feb;104(2):602-11. doi: 10.1002/jps.24259. Epub 2014 Nov 21....case, a buffer exchange can be performed using a desalting column or an ultrafiltration concentrator. As ...suggests, you may also concentrate your prep to the desired concentration using these columns. If you are conjugating...

...take care when using this method.      Fig 2:  A, The polyhistidine tag is a string of histidine... Bio/Technology. 1988; 6: 1321–1325. Janknecht R, de Martynoff G, Lou J, Hipskind RA, Nordheim A, Stunnenberg... for nonspecific binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins... I place the tag and how many do I need? When designing a plasmid containing a polyhistidine tag, you’...

...efficient knockout (KO) of genes in immune cells; (2) is not immunogenic, a necessity for in vivo studies...single gene KO and pooled screening approaches; (4) encodes a Thy1.1 selectable marker to facilitate the isolation...assess T cell responses and clonal dynamics in lymph nodes (LN) and tumor cells. Figure used with permission...also reveal novel pathways. The FITS deposit also includes pLX_305_Ovalbumin, a plasmid that enables introduction...

...some rockin’ newly designed Addgene swag. Good luck and happy naming!   Fig. 2: The mascot trio!...

...of ~75% in response to the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled...didn’t need any added cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a variant...Maria were encouraged by previous reports that described magnetic responses of cryptochrome proteins or...

...retain their individual activities when separated (2), and that DBD and TAs from different systems could... Enzymol. 1983;100:293-308. PubMed PMID: 6312261. 2. Keegan, L., Gill, G., and Ptashne, M. Separation ...activation. Including a prey-only control also provides a baseline level of reporter activation. Vary ...

...faster when using a dual guide system.   Fig. 2: Size screening for large deletion events.   ... Biol 17: https://doi.org/10.1186/s13059-016-1012-2 Find more CRISPR Resources at Addgene: Read our CRISPRGuide...screening by PCR may be the way to go. Design a PCR to flank your desired edit site and run the PCR product...nucleotide along with your desired edit. This silent mutation should be designed solely for restriction enzyme... updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells... your cells were appropriately edited for your desired mutation - insertion, deletion, or site-directed...know a few things about your target cells and the desired edits. First, you will need to know what the ploidy...

...sequence is unique compared to the rest of the genome.  2) The target is present immediately upstream of a Protospacer...utilized allowing for PAM flexibility. Fig. 2: Prime editor engaging target DNA. The reverse transcriptase...vectors and design your sgRNA targeting sequence using one of the many freely available sgRNA design programs...many cases several exons’ worth of DNA. Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single...Cas9 localizes the deaminase to where the edit is desired, and it performs the programmed edit within the...flexibility. A consideration with this tool is that the desired edit will be performed on all relevant bases within...system utilizes an RNA template containing the desired edit adjoined to the sgRNA, a reverse transcriptase...

...blood are reprogrammed immediately after collection [2]. PBMCs display unique epigenetic signatures that ...PMID: 27882265. PubMed Central PMCID: PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced ...iPSCs. The generation of human iPSCs (hiPSCs) now provides an opportunity to develop and use patient-specific...for reprogramming purposes. In this post, I’ll describe an episomal vector system that increases reprogramming...the CytoTune™-iPS 2.1 Sendai Reprogramming Kit designed to generate clinical grade hiPSCs is sold at $11,000...replication, OriP and the EBNA-1 sequence that encodes for the DNA binding protein EBNA-1 [7]. These two...reported efforts. The pCXLE episomal system thus provides a practical way to generate transgene-free, virus-free...

...ligands (Figure 2). Biosensors, which include calcium and voltage sensors, are the broadest category of viral...’re delivering their payload to.   Figure 2: Numbers of AAV tools, by category, available from...site, and titer. Addgene’s Viral Vector Service Besides being complex to use, viral vectors are also difficult... for vectors in the “controls” category, which includes things like viral vectors that deliver GFP. ...most requested serotypes (Figure 4), our catalog includes vectors in a variety of serotypes, including the...early engagement with UPenn to our launch of the redesigned Data Hub in 2022. It’s influenced the way we ...viral vector educational resources, from eBooks to guides to protocols, which was why I was so excited to...

...whether the results include knockout data.    Figure 2: Search options in the Antibody Data Hub. Data reports...antibody worked under the conditions described above, and a short description of how the antibody performed....the submitted application, along with a brief description of the experiment. If that data was included ...ELISA — but in all cases, it contains a detailed description of the experimental conditions.     Figure 4...The submission process is easy, with simple, descriptive fields, drop-down boxes when appropriate, and...

...Information Science and Technology 60.4 (2009): 858-862. 2. Van Noorden, Richard. "Interdisciplinary research...Librarianship and Scholarly Communication 2.2 (2014): 2. Additional Resources on the Addgene Blog 6 Tools.... After months, or years, of grueling effort to design, fund, and conduct a research project, the vast...provide more than enough time for researchers to describe their science, and for viewers to decide if reading...is of interest. In his research, Scott Spicer concludes that “… the emergence of the video abstract genre...websites). WeShareScience is a social platform that provides links to the videos, keeping the copyright and...

...prompt you to log in or create an account.  Figure 2: The new deposit materials bar New menu Most common...and the Addgene Help Center. A big part of this redesign was pulling all of our science options into a ...Addgene’s website easier to navigate, thanks to design choices informed by Web Content Accessibility Guidelines...word for it — check out all the new features and designs addgene.org! And when you’re finished, feel free...

...PMID: 24336571. PubMed Central PMCID: PMC4089965. 2. Sanjana, Neville E., Ophir Shalem, and Feng Zhang...Feng Zhang’s lab at the Broad Institute and was designed and cloned by Neville Sanjana. This fantastic ...your gRNA of interest to mammalian cells. It was designed to have increased lentiviral titer as compared...sampling of the research it has already been used for includes work probing mTOR signaling in cancer cells, deciphering...