...more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising monoclonal antibody...an amide group added to its C-terminus post-translationally, which turned out to be critical for antibody...

...does not greatly alter the size of the tag.   Table 1: Common protein tags Tag Epitope Mass (kDa) Function... conditions FLAG DYKDDDD or DYKDDDDK or DYKDDDK 1 Affinity and Purification Good for antibody-based ...usually smallish peptides incorporated into a translated protein. As depicted in the accompanying cartoon...peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant proteins...

...dark affinity range (130nM to 4.7µM for iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this... August 20th; 14(1). TRICK: A method for visualizing the first round of translation in living cells ...light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2). In the dark, this peptide is caged by the...Figure 2). They are all available now at Addgene. 1 Lungu et al., Chem Biol. 2012 Apr 20;19(4):507-17....Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; 112(1):112-7. doi: 10.1073/pnas. CRISPR-Cas9 optogenetic...for a variety of experimental purposes including (1) single or multiple, constitutive or inducible, gene...the years. The authors optimized these systems by: 1) decreasing the number of plasmids required for each...

...fragments of these transcripts. I remember when small RNA was just something we ran off the bottom of ...viruses - 2 cas systems regulate at the DNA level and 1 at the RNA level. Udi Qimron from Tel Aviv University...’t give tRNAs much thought. High throughput transcriptome sequencing methods including work done by the...hypothesized that life started with RNA and a simple cell membrane. His recent work shows that they are making fairly...knew…I had barely heard of these). These single stranded, covalently closed RNAs seem to be conserved in...

...single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP) and...genOway press release for more information. References 1. Atasoy, D., Y. Aponte, H. H. Su, and S. M. Sternson...concentration of FLEx vectors guarantees that most cells are transfected with all labeling plasmids and...PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart. 1998. “Improved Properties of ... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ... has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart 1998). FLPe’s performance was further...in FLPO, which shows similar efficiency to Cre (Kranz et al. 2010). These differences in recombinase efficiency...

...Addgene's founders Podcast breakdown Intro Chapter 1 - Who are Addgene's Founders? Chapter 2 - The process...Chapter 3 - Addgene as a leader in standardized tech transfer and the adoption of the UBMTA Chapter 4 - Developing...ensuring that success Chapter 6 - Developing Addgene’s Brand and culture Chapter 7 - Building Addgene as a global...

...Figure 1). Clustered events are called a population.                             Figure 1: While...conceptualize where the populations are, try drawing a quadrant over the plot that divides it into four, using...different populations by imagining (or drawing) a quadrant over the dot plot. Created with biorender.com....Biorender! While a helpful visualization tool, a quadrant will often be too clean for real-world data. Most...and debris. The side scatter roughly measures granularity, a proxy for nucleic complexity of the cell.  ...these populations, I suggest using the imaginary quadrant to help (Figure 5). Even though this real-world...real-world data is far messier than biorender plots, quadrants can still help me as a reader if I'm struggling...

...deficient in DNA cutting activity to a transcriptional activation domain (Fig 1). Two ways to use CRISPRa for... (2016) Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems. PLoS...promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides...been significant work on constructing Cas9 transcriptional activators. These constructs allow for the ...picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the...Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12:326–328 . https...Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

...Spaghetti Monster probes at Addgene Uses of smFPs 1. Multi-channel connectomic tracers Fluorescent proteins... expanded use of smFPs in the future. References 1. Viswanathan, Sarada, et al. "High-performance probes...ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with facile ...

...know that most cephalopods die after they lay their 1 clutch of eggs? Propagation is thus quite difficult...organism blog posts: Five Popular Model Organisms, Part 1 Five Popular Model Organisms, Part 2 ...species. Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues...Phenotype thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific...evolution because electric organs arose in different branches of the evolutionary tree at least 6-7 times and...mushrooms) but there are over 1,000 species. No transformation techniques are available yet. Polistes (Amy... a system using novel vesicles to efficiently transform these cells which don’t like foreign DNA at all...

...folding, and deal with sub-optimal codon usage (Table 1) A few mutations are common to all or most expression...curated list of Bacterial Expression Systems Table 1: E. coli expression strains  Note: All strains are...specialized strains are available to confer greater transcriptional control, assist with proper protein folding...harboring this mutation are deficient in outer membrane protease VII, which reduces proteolysis of the...Lacks functional RNaseE which results in longer transcript half-life F- ompT lon hsdSB (rB- mB-) gal dcm...Chloramphenicol (pRARE) Good for “universal” translation; contains 7 additional tRNAs for rare codons ...pLemo (CamR) Expression of toxic, insoluble, or membrane proteins  T7 Express   IPTG-inducible expression...

...sequence on either side of the desired mutation (usually 1-3 mismatched bases) is sufficient for your primers...researcher at Cornell University.       References 1. Breslauer, Kenneth J., et al. "Predicting DNA duplex...homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the...products of the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data...final PCR products are pieces of linear, double stranded DNA with homologous ends. These homologous ends...the adenosine of the GATC recognition site. Transformation: After the PCR reaction, no ligation is required...

...$47,476 and the date for implementation was set for Dec 1 2016. In order to be exempted from the need to pay... these institutions (populations potentially from 1 to 5761, according to the NSF data) and therefore ...about? In March 2014, the White House issued a memorandum to the Secretary of Labor to update the FLSA,...postdocs in the final ruling was, for a lot of us, frankly quite surprising. Consortia of higher education...issued, a joint blog post by Director of the NIH Francis Collins and Secretary of Labor Thomas Perez, Fair...exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral positions... of postdocs at different points on this salary range. We have been able to gather data on changes to ...

...possible without double-strand breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime...demonstrated ~20-50% efficiency in HEK293T cells with 1-10% indels. Since then, further innovations have continued...Engineering. https://doi.org/10.1038/s41551-024-01227-1 Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward... non-edited strand, they can cause the cell to remake that strand using the edited strand as the template...the nicked target strand. The initial result is a heteroduplex, with overlapping strands of edited and unedited...Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research, the Liu...the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited ...

...different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure 1: Transformation... differences between transformation, transduction, conjugation, and transfection Resources on Addgene.org...Transformation introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation...bacterial transformation of chemically and electrocompetent cells is minimal (picogram range). It can ...plasmids into mammalian cells? Use transfection grade DNA. Transfection is the process of inserting an ...without commercially available kits and DNA for transformation doesn’t need to be as pure as for other uses...scientists to clone from a smaller amount of DNA (ng range). To allow efficient and flawless cloning it is ...

...forward to another great year of sharing!   References 1. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout... name suggests, lentiCRISPR v2 is a lentiviral transfer vector used to deliver both Cas9 and your gRNA...metalloproteinase-9 secretion, and identifying lysine methyltransferase substrates. We’re incredibly happy that lentiCRISPR...

...is here to help. Contact us at [email protected] or +1 617-225-9000, or check out our MTA reference pages...Uniform Biological Material Transfer Agreement (UBMTA) for its material transfers. The UBMTA was drafted by...organizations Assurance that recipient may not use the material for a commercial purpose Assurance that recipient... has time for paperwork? Here’s why a Material Transfer Agreement (MTA) matters: Science thrives on collaboration...universities and institutions require them for material transfers. When Addgene entered the plasmid sharing space...receiving institution. Therefore, many technology transfer officers are familiar with the UBMTA and are happy...  material transfer agreement...

...’ve come to the right place. Get started with PGC-1 plasmids from Addgene. References: Skeletal muscle...PGC-1a1, a transcriptional coactivator induced in skeletal muscle by exercise and endurance, protects ...have reported a mechanism based on studies of transgenic mice that helps to explain how it works. The ... skeletal muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into...

... successful after their training.   Figure 1: Professional Development Hub Workshop Attendees and...ample resources and a positive perspective on the transition to a post-academic career. This training was ... inadequate job of supporting science trainee transition into diverse, successful careers.  We’re not ...components of predoctoral and postdoctoral training. A grant from the National Institute of General Medical Sciences...

... specific post-transcriptional regulation of endogenous RNA. J Biol Eng. 2014 Mar 1;8(1):7. doi: 10.1186...ability to direct enzymes – from nucleases to transcription factors – to specific sequences of DNA has become...Biological Engineering in March is fused to a translational repressor. In principle, however, designer PUF...