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...Principles and Clinical Application. Clinical and Developmental Immunology 2012:1–15 . https://doi.org/10.1155...the other (Sandomenico et al., 2020). Figure 1: Comparison between the IgG antibody and scFv. ...intensity of a tagged protein. SunTag has two components: 1) a protein of interest that’s tagged with 10-24 copies...without disulfide bonds, made by molecular evolution 1 1Edited by I. A. Wilson. Journal of Molecular Biology...led to their application for basic and translational research.  Basic research applications of scFvs: ...blood clearance, which is useful for imaging applications; better tissue penetration which is useful for... for therapeutic and imaging applications; and reduced immunogenicity when administered in vivo due to...

...glassware should be treated with a DEPC solution for 1 hour, and autoclaved to remove residual DEPC. Alternatively...more large, expensive ultracentrifuge.  Figure 1: An outline of the different steps in RNA extraction...extraction: twenty-something years on. Nature Protocols 1:581–585. https://doi.org/10.1038/nprot.2006.83 Additional...of DNA that can potentially complicate results of a downstream application such as quantitative PCR to...easier. Recently, we published a blog on DNA purification without a kit that outlined several reasons ...take care whenever doing any type of DNA or RNA purification to avoid contamination, take extra care when..., that isn’t the case for RNA extraction and purification. There is one simple method that works, and ...

...mutation site, indicating that repair proceeded through NHEJ rather than HDR. Figure 1: Homology-directed... employing HDR. Ma et al. moved forward with gRNA 1 to conduct microinjection experiments in S phase zygotes...instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies of...thus allowing repair to take place before DNA replication. Indeed, they observed that 41/42 embryos were...attributable to CRISPR editing. Conclusions & implications One striking feature of this paper is the failure... need to be much, much lower before clinical applications could be considered. It is important to consider...experiments are needed before we can consider clinical applications for germline editing. Although this paper represents...

...various cyan fluorescent proteins is depicted in figure 1. Of note, the photobleaching rate does not depend ...to be used with the fluorescent protein. In figure 1 we provide an example of such a comparison between...the variants is comparable (lower panel of figure 1). To avoid changes in excitation power, it is important... localized fusion proteins and do not require dedicated plasmids or constructs. It should be noted that...reached at high power may not translate to real applications in which far less power is used. For example...insight into the photostability in the intended application (Goedhart et al, 2012). The third issue relates...Selecting a photostable protein The issues with quantification of photobleaching rates can be summarized as...

...succumb to the toxicity of CcdB.   Figure 1: Image from Bernard, P., et al. showing the pKIL 18...submit a guest blog entry of your own! References 1. Bernard, P. “Positive Selection of Recombinant DNA... plasmid ccdB killer gene”. Gene. 1994 Oct 11;148(1):71-4. PubMed PMID: 7926841. 4. Bernard, P., et al...time-consuming aspects of traditional cloning is the identification of clones that actually contain your insert...technology. CcdB: A potent toxin... The ccdB gene, located on the F sex factor plasmid of E. coli, is part...enzyme-mediated disruption of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful...which can then be used for downstream cloning applications. While these two strains ultimately perform ...

...using the format indicated below and pool-clone them into the pAWp28 storage vector (Figure 1): Forward oligo..., including those found at Addgene! Figure 1: Strategy for assembling the barcoded gRNA library... He can be contacted at [email protected].   References 1. Cheng, Allen A., Huiming Ding, and Timothy K. Lu....realization of a variety of perturbations and applications in functional genomics, cell reprogramming, ...CRISPR/Optogentics Tools Resources on Addgene.org Catch up on Your CRISPR Background with Our CRISPR Guide...

...Transfer Vectors   Expressing your gene of interest pMKO.1 GFP (Plasmid #10676) Retroviral vector for cloning...GFP expression.  Cloning and expressing RNAi pMKO.1 puro (Plasmid #8452) RNA interference (RNAi) is an...information and a detailed protocol on how to use the pLKO.1 vector can be found here. Cre-Lox technology Puro....-humanized (Plasmid #44246) Expression of a catalytically inactive (dead), human codon-optimized Cas9 ...be used to construct complex lentiviruses for modification of mammalian cells with a single viral infection...Gammaretroviral Vectors: Biology, Technology and Application. Viruses. 2011. PubMed PMID: 21994751. PubMed...

... experimented with a group mentoring format where 1 mentor met with a group of mentees to get more “bang...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an...get together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work. If ...This is a chance to practice leadership and communication skills. The leader also provides materials such...

...neuron which generates an action potential (Figure 1). This light-induced excitation can take place in ...channelrhodopsin activation to a small volume of brain tissue (~1 mm3). Current channelrhodopsins also need high-intensity...fiber-optic cables into the brain. Figure 1: With no blue light, the channelrhodopsin remains ...tegmental area (VTA) of the brain. Since the VTA is located in the midbrain, fiber-optic cannulas were still...right secondary motor cortex (M2). Since M2 is located on the surface of the brain, to deliver light to...

...point mutations with unc- 119 + selection." MicroPublication 1 (2018): 2–3. Wang, Han, et al. "cGAL, a temperature-robust...Zhang et al., 2015). In this system a modified TIR-1 protein can degrade degron-tagged proteins in an auxin...splitting cGAL4 in two and binding each half to a gp41-1-N-intein. Split inteins are protein domains that naturally...the OpenWorm website. microPublication microPublication is a unique publication for the worm community...into larger publications and would otherwise be lost in lab notebooks forever. microPublications are peer-reviewed...aims and approach of CeNGEN, check out their publication.   3’UTRome The 3’UTRs of mRNA are important ...normally one figure and can be negative and/or even replicated studies to show reproducible results. This a ...

...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...

...targeted expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity of the...used four different design strategies: Strategy #1 (ProA): Create promoters with sequences upstream of...active in retinal cell types in mice. Figure 1: The four strategies used to design synthetic promoters...channelrhodopsin, fused to a fluorescent GFP marker (CatCH-GFP). The lab deposited AAV plasmids containing ...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression... promoters expressed in. The lab tested this application using one promoter to drive expression of Cre-mCherry...

...nucleotides are represented equally, that’s only a 1/16 chance of having a PAM at a given site. While it... NYN/NRN NYN is less effective than NRN Table 1 - Summary of PAM-flexible Cas enzymes Are we truly...editable with CRISPR technology.     Fig. 1: SpCas9 has sparse PAM coverage in the human genome...flexibility in the location of the cut and subsequent edit. In these applications, location takes priority...make a choice about what is your top priority: location or efficiency. If you are just trying to shred...still leave you choosing between efficiency and location.   Nearly PAM-less editing   The Kleinstiver lab...in double stranded cleavage and base editing applications, with high editing capacity in both contexts...

..., check out Addgene's CRISPR Guide. Figure 1: Like this jar of jellybeans, a pooled CRISPR library... selecting a CRISPR library for your experiments. 1.  What species are your cells derived from? Currently...a different gene. Libraries may be available in a 1-plasmid system, in which Cas9 is included on the gRNA-containing... unique compared to the rest of the genome and located immediately 5’ to a protospacer adjacent motif ...coli, and T. gondii genes.  2) What genetic modification are you trying to make? Addgene carries CRISPR...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral... As such, sequencing CRISPR libraries after amplification and after a screen requires the use of next-...

...backbones for shRNA expression, such as pLKO.1 puro and pLKO.1 – TRC cloning vector. Lentiviral Plasmids ...they were added to the repository, including beta-catenin reporter M50 Super 8x TOPFlash, scramble shRNA ... GCaMP6 series of genetically encoded calcium indicators were the hot technology of the year in our collection...

...unlimited supply of identical antibodies (See Fig 1.). rAbs are a relatively new technology that takes...conveniently modified and engineered.   Fig. 1: Antibody production methods    NeuroMab and others... the signal. If the exposure time is long (e.g. > 1/7s) then the signal is weak and vice versa for a strong...the best, or even which one will work for your application? Like many scientists, I have spent hours sifting...produced from a new set of animals), and wrong application (different experiments/conditions can change ...differently over time), compatible with the desired applications (such as immunohistochemistry (IHC) or western...colleagues! Dr. Wu and his lab members (like me!) are dedicated to creating a public and accessible database for...

...-public settings. Ask over coffee.  Have regular 1:1 meetings with team members (don’t become known for...Scientists series will focus on an aspect of communication especially important for a manager – getting... Creating a culture of excellent, effective communication between all members of a team. As described ... opinions in meetings, via email, at formal communications events or at informal gatherings, like at lunch...

...about HIV cure research and science communication.     References 1. Gaspar, H. B., S. Howe, and A. J. ...reconstitution with a normally functioning T cell repertoire (1). In this example, it was possible to transduce the...significant improvements in vision over a period of 1 to 3 years – the treatment appears to be both safe...affinity-purified receptor-targeted viral vectors." Nature communications 6 (2015): ncomms7246. PubMed PMID: 25665714...our AAV Production Protocol Check out our AAV Purification Protocol ...

...and accessible education in gene editing and synthetic biology. Nature Communications, 15(1), 6563. https...more.  Figure 1: Workflow for cell-surface protein labeling and identification. Reproduced from Wu...with a membrane-impermeable biotin substrate (Figure 1). Biotinylated proteins were isolated, identified ...564–571. https://doi.org/10.1038/s41586-022-05682-1 Zhang, Y., et al. (2025). Probing condensate microenvironments...cell mitochondrial transfer tracker Red calcium indicators PinkyCaMP and FRCaMPi Micropeptide killswitch...the Cre-tagged mitochondria, Cre is released, translocates to the nuclease, and excises the DsRed-STOP ...doi.org/10.1073/pnas.0510511103      Red calcium indicators PinkyCaMP and FRCaMPi By Mike Lacy Many popular...

...depend on your intended application and available resources.     Figure 1: Table summarizing the... a good option for applications like immunohistochemistry on human tissues, when (1) other strategies ...expected in another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...validation: A view from the mountains. New Biotechnol 45:1–8. https://doi.org/10.1016/j.nbt.2018.08.002  Uhlen...intended application - Remember, just because your antibody performs as expected in one application does ...strategies for antibody validation in different applications. Here, I want to highlight the general suggestions...intentionally generalizable and not specific to certain applications or antibody targets. That said, not every strategy...