...purple) and Gram negative (red/pink) staining. Image credit: Mark Perkins https://www.flickr.com/photos/occbio...charged iodine is added, it interacts with the CV+, creating a complex within the peptidoglycan layer, staining...peptidoglycan layer, trapping the large dye complex created by the interaction of CV+ and I- in the thick multilayered...positive and negative classification system is incredibly helpful in visualizing differentiations between...What is a plasmid? Plasmids 101: Blue-white screeningPlasmids 101: Common lab E. Coli strains  ...

...resulting in a decrease in luciferase activity. This experimental protocol can readily screen miRNAs for ...Peak emission (nm) Size (kDa) Substrate Cofactors Secreted Luminous shrimp(Oplophorus gracilirostris) 455...cell membrane and deliver the substrate; however, secreted luciferase or the use of alternative substrate...pathways can be investigated in a high-throughput screen using luciferase activity as a measure of signaling...

...the potential for increased speed and efficiency through which knowledge is created and shared. Can you...platform. Expanding further beyond the US and increasing Addgene’s international presence would potentially...information, making it available to everyone, that creates more breakthrough and innovation. With any kind...science regulatory law. My role at Suffolk is to help create new programmatic content as well as help our students...

...that approach seems to create competition for your own spot. True! But you create a network of friends,... and simplify access to academic resources, we created STEAMid, for a nationwide database of  STEAM internships...with your question(s). This precautionary step increases the likelihood of acceptance to the internship... following graduation. While these suggestions increase your competitive edge, they alone, are no guarantee...

...sequence desired. This technology allows you to create a pair of designer proteins that will introduce... transfection or electroporation. All you do to create a protein that binds those 15 base pairs is take...genome editing tools allow you to do is greatly increase the efficiency of the genome editing process. ...for investigators without a lot of expertise to create zinc finger nucleases that will bind desired sequences...in terms of the target site. Also, any time you create double-strand breaks, there is a possibility of...

...address this need, the Khavari lab at Stanford created the RNA-protein interaction detection (RaPID) method...RaPID. Ramanathan et al. also tweaked BirA* to increase its rate of biotinylation. The improved enzyme...in neural progenitor cells. Knock-down of QKI decreased Zika viral RNA levels by 90%, suggesting that ...Hyperferritinemia-Cataract Syndrome (HHCS), a disease characterized by increased iron serum levels and early-onset cataracts, is...sequences and that loss of binding correlated with increased serum iron levels seen in HHCS patients. These...

...antibody and a ≥100-fold increase in signal over intrinsic fluorescence. Increased signal is likely due to...is a ~150 amino acid, single domain antibody. To create their RANbodies, Yamagata et al used previously... RANbodies are expressed in 293T cells and are secreted from cells due to their IgGκ signal peptide sequence...them. HRP RANbody staining results in a ≥10-fold increase in signal over staining with a primary/ labeled...

...also underwent editing through the NHEJ pathway. Created with BioRender.com. In later characterization...current tools, thus quieting some fears about the creation of “designer babies.” The differences in editing...disorders, pre-implantation genetic diagnosis (PGD) screens embryos to find those that do not carry a given.... claim that CRISPR would still be helpful in increasing viable embryos, especially in the case of older...still be worthwhile for a given disease if it increased the percentage of disease allele-free embryos ...

...scaffold to enhance complex assembly, thereby decreasing the amount of background fluorescence from unbound...CRISPR to label multiple loci in distinct colors. To create a colorful Cas9 toolbox, Ma et al. turned to SpCas9...was fused to a different fluorescent protein to create three distinct colors. The specificity of these...distinct hairpin sequences produces white light. Created with BioRender.com. With this rainbow of colors...combination, for example, with a far-red FP, would increase the available colors to 15! They also envision...

...visibility is increasing internationally, thanks to evolving outreach tactics, increasing business development...they see the value in depositing and they are increasingly seeking us out on their own to share reagents... We were and remain ahead of most companies in creating a welcoming, inclusive workplace for everyone....about their dedication, excellent intentions, or incredible drive to succeed.  Thanks to all the Addgenies...

...Institute has created hundreds of thousands of plasmids, using both mass production genome wide screens and the...vector. The time it takes to make a plasmid could increase dramatically if you are piecing together several...at Addgene, the consumables cost per stab has increased 50% since 2019, while overall inflation of USD...equal to the $155 spent on reagents. Five ways to decrease plasmid production time and cost If you’re short...

...Foundation, and Stanford University—that harnesses the creative power of sex and gender analysis for innovation... in research Governments, universities, and, increasingly, corporations in the US and Western Europe have...decades: “Fix the Numbers of Women” focuses on increasing the numbers of women participating in science...gender analysis and 2) provides case studies with concrete examples of how gender analysis leads to discovery... Environment—a project aimed at harnessing the creative power of sex and gender analysis for innovation...

...these demands? Are there other solutions, such as increasing numbers in Master’s level programs? What would...Maryland College Park, is a strong supporter of increasing the number of postdoctoral scientists at University...School. He has continually promoted the cause of increasing data collection about the scientific workforce... Boston 2015 where we hope to begin developing concrete solutions to the problems looming over the academic...School. In the lab, he uses synthetic biology to create tools to better understand human disease.  Outside...

...particularly given that this has precipitated increases to the NIH recommended pay scale – used by many...while this is a move in the right direction, the increase notably still does not meet the suggested postdoc...this area, keeping the pressure on for further increases will be challenging, but will be aided by the ... new strategies designed to cope with current increases. These two rulings along with progress over the...School. In the lab, he uses synthetic biology to create tools to better understand human disease.  Outside...

...grandsons that decreased their likelihood of death from cardiovascular disease but increased their risk of... DNA methylation patterns, either large increases or decreases. Hypermethylation of CpG islands in the... units of the FRET pair together and causes an increase in the FRET signal. Subsequent demethylation separates...

...amount of agarose/DMEM should be proportionately increased). For some cells, add a drop of serum to aid in...up, most will eventually grow within a week. We screen the wells in groups as they become confluent, taking...important to have a functional assay in place to screen your clones. Depending on the protein, we have ...SH-SY5Y cells, as these cells did not seem to form discrete colonies but constantly migrated away from each...either initially, or using secondary biological screens. For example, for cancer researchers, if growth...

... to its final destination. As an example, most secreted proteins are produced in the endoplasmid reticulum...golgi and then transported in vesicles via the secretory pathway to their subcellular or extracellular ... they are finally degraded (2, 3). Once you've created your fusion protein, it's important to validate... to make any fluorescent protein constructs you create in your own work available to the Addgene community...Edition. 3.  CureFFI.org. Cell Biology 04: The Secretory Pathway. 4. Dunn, Kenneth W., Malgorzata M. Kamocka...

...needs to be compensated out of APC-Cy7. After increasing the compensation values for APC against APC-Cy7...to be shifted towards the x-axis, so you would increase the compensation value for APC. If the population... to be shifted away from the x-axis, you would decrease the compensation value.   Figure 1: Compensation...population needs to be shifted towards an axis, increase the compensation value, whereas if it the population...population needs to be shifted away from an axis, decrease the compensation value. If neither of these helps...

...detectors for all fluorophores. This allows for the creation of much larger panels, as fluorophores that cannot...they don’t “wash out” the rest of the panel and create spreading error. Conversely, markers with low expression...should not be paired with these dyes, as this would create difficulty when trying to resolve those populations...signature upon antibody binding, leading to discrepancies during unmixing. Additionally, it is critical...spectral flow cytometry has the ability to generate incredible amounts of data from a single experiment. Although...

... aaRSs in these processes. To create the antibody toolbox, they screened antibody candidates using a synthetic...fourteen aminoacyl tRNA synthetases (aaRS) have been created, characterized, and validated by the Gräslund lab...cross-reactivity, antibody candidates underwent affinity screening to select for slow off-rate binders. The best ...pathways By Gabrielle Clouse Claudia Vickers’ lab has created a novel nanocompartment for transporting proteins...

...transfer plasmid at 30 °C instead of 37 °C and then screen for ITR recombination with a SmaI or XmaI restriction...duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary. To save time...mL of my AAV transfer plasmid cultures and then screen with SmaI digests. I freeze the bacterial pellet...how iodixanol purifications works, shows how to create the iodixanol gradient layers, and has some great...master iodixanol AAV purification. If you’re new to creating gradients, practice making the iodixanol layers...

...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the insertion...obtained if any single component was omitted. Screening for successful Golden Gate Assembly All Golden..., Table 1). These improvements (5- to 12-fold increases in the number of transformants while maintaining...to faster assembly workflows that require less screening to confirm the accuracy of assembly. The fidelity...test the limits of DNA assembly approaches with increasingly complex experimental conditions. The ability...

... deletions or homology-directed repair (HDR) to create precise genome edits. But have you heard of another...depositor Takashi Yamamoto’s lab has harnessed MMEJ to create a new method for CRISPR gene knock-in, termed PITCh...PITCh plasmids, GFP knock-in cell lines can be created in about a month and a half, without the need for...PITCh-gRNA, and the locus-specifc gRNA. The PITCh-gRNA creates two DSBs in the PITCh plasmid to release the cassette... alter expression of the gene it follows. For increased versatility, it would be advantageous to adapt...

... or the creation of bicistronic or multicistronic vectors. Unlike promoters which will create unique mRNA...resistance gene. These reporters allow you to easily screen or select for cells that are expressing your gene...interest at high levels. Unlike vectors that express screenable or selectable markers from a unique promoter,...now download Addgene's new eBook! Our goal was to create a one-stop reference guide for plasmids. We've ...

...researchers saw an 11-fold increase in mRNA levels after 2 hr and a 400-fold increase after 30 hr. The system...(CIB1) proteins. The goal of both groups was to create a system that would use light to turn on and off...domain to each of the Cas9 fragments, the authors created a photoactive Cas9 tool, as shown in the figure...binding and cleavage. This technique was applied to create photoswitchable spCas9, dCas9, and S. aureus Cas9...Gilgenast TG, Gong W, Beagan JA, Raj A, Phillips-Cremins JE (2019) LADL: light-activated dynamic looping...

...Komagataella phaffii (a.k.a  Pichia pastoris) because it secretes fewer of its own proteins, making the purification...production of Fab fragments where variable chains are secreted into the periplasmic space to be oxidized and ...incubation time to maintain cell viability and increase protein production (Schwarz et. al., 2020). Harvest...   Figure 1: The steps of rAb production. Created with biorender.com   Another method of harvesting...contaminating bands or a few strong ones, consider increasing the number of washes performed during your harvest...

... 2021). In fact, completion of Provost’s CURE increased students’ confidence in their research capabilities...projects have also been proposed as a mechanism for increasing diversity and inclusion in STEM disciplines (... Brownell, 2014; Duboue et al., 2022), as they increase student interest in scientific career paths while... Provost believes these networking experiences increase student enthusiasm, and research backs up that...undergraduate research experiences are a “CURE” that increases engagement by students and teachers. FEMS Microbiology...

...the templated DNA. Check out our blog post on increasing HDR efficiency to read more about optimizing ...pathways have been shown to be efficacious for increasing knock-in events (Yang et al., Int J Mol Sci)....culture media prior to and after Cas9 introduction increases HDR, just make sure to remove the inhibitors within...Early HR factors fused to Cas9 have been shown to increase HDR frequency by committing a break to performing...reading up on validating genome edits to find screening strategies for your experiment. Also learn about...

...large number of constructs for high-throughput screening of PPIs. For example, Bischof et al. (2018) generated...generated an open reading frame (ORF) library to screen PPIs among 450 transcription factors in live fruit...organisms lags behind. BiFC can also be used to screen for small-molecule inhibitors of a particular PPI...Here, one of the endogenous PIPs is expressed at increasing levels alongside your BiFC constructs. If your...co-expressing one of the endogenous PIPs should decrease the BiFC signal in a dose-dependent manner. This...

...CRISPR, and the challenges of science becoming increasingly politicized. It would be nearly impossible for...all of these things contributed to the steadily increasing distrust the general public has in science and...self-promotion, and personality is playing an increasingly important role in science. The science produced...reserved Franklins and Prashers will find it increasingly difficult to compete (for funding, etc.) with...chapter on deep learning addresses the dilemma of creating a technology that could eventually replace scientists...

...newest line of defense against bacteria. Created with BioRender.com.   The problem of antibiotic...may also induce cell death. CRISPR also enables increased specificity - rather than targeting a process ...mouse skin colonization model, phagemid treatment decreased the proportion of virulent S. aureus from 50% ...death. Image from Beisel et al., licensed under a Creative Commons Attribution 4.0 International License....rendering the population sensitive to antibiotics. Created with BioRender.com. Phages could be used to...

... shalt create community to help Involve family. If you don’t have local family to help, create “fake” ... on the Society for Laboratory Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work... to church, join a club, meet the neighbors and create a network of support to help with elder care, carpooling...partner – no kids. Get a massage – no guilt. Have recreational time with your friends – no spouses or kids....

...is possible with efficiency >70%. CRMAGE is an incredibly fast protocol, with only 5 hours incubation time...system that combines recombineering with CRISPR to create a system for scarless, iterative genome engineering... pathways within this genus, Huimin Zhao’s lab created two “pCRISPomyces” systems for use in Streptomyces...CRISPR in bacteria, they also led early efforts creating tools for CRISPRi and CRISPRa (Bikard et al., ... in Mycobacterial species, Sarah Fortune’s lab screened 11 other Cas9 orthologs to identify a more efficient... used to engineer the bee gut microbiome. They created a modular system using a broad host range plasmid...

...to solve this problem. This tool was initially created to support the CRISPR analysis needs of Synthego... sgRNAs. Since multiplexing is commonly used to create functional knockouts or large deletions, the ability...optimizing and no complicated steps to learn. For increased flexibility and scalability, the ICE software ...editing analysis Once the analysis is complete, a new screen appears with a graphical representation of the ...Data button on the bottom right of the analysis screen. ICE analysis details Contributions   The Contributions...

...careers. External collaborations are becoming increasingly important as more complex approaches to scientific...seminar to connect the imaging community and to create a platform for discussions and potential collaborations...postdoctoral work there are loads of opportunities to create communities, platforms, and to be a part of projects...your ideas and don’t be afraid to make something concrete happen. You may have more willing collaborators...make sure to share them with a short note - this creates a thoughtful way of interacting and is much more...

...fluorescent protein can be truncated to increase the FRET efficiency. Creating fusions at the N- and C- termini...double the size of the protein. In addition, it increases avidity because it doubles the number of binding...non-aliphatic amino acids. However, long linkers increase the chance that a fusion protein is cleaved by...Sheridan, Douglas L., et al. "A new way to rapidly create functional, fluorescent fusion proteins: random...

...for quantitative imaging, Ming Hammond’s group created Spinach2 using site-directed mutagenesis of Spinach...was cloned into bacterial expression vectors to create a library. Bacteria were transformed, and then ...for in vivo studies. Dimerization of Broccoli increases its fluorescence by about 1.8-fold, and may be...for greater contact with its RNA aptamer. This increased interaction between Corn and its small molecule... to TO1-biotin. Two of these variants exhibit increased brightness compared to Mango, and all are photostable...

... ligase The Roux Lab in 2016 optimized BioID to create BioID2, a smaller and improved promiscuous biotin...was its low sensitivity. Sensitivity could be increased with higher expression levels but for many fusion...until they identified a mutant with significantly increased sensitivity that did not affect the function or...miniTurbo were developed in the Ting Lab in 2018 to increase the speed of proximity labeling by promiscuous... BASU exhibits 1,000 fold faster kinetics and increases the signal to noise ratio 30-fold compared to ...

... used for a variety of research purposes, like creating stable cell lines, delivering CRISPR/Cas9 to cells...vector with an integrase-deficient viral vector. Created with BioRender.com. What are integrase-deficient... episomal forms of non-integrating transgenes. Created with BioRender.com; adapted from Banasik and McCray...regions of the genome (Schröder et al., 2002), thus increasing the risk for insertional mutagenesis. This integration...remember: You can never trust a lentivirus with secrets... Because they tend to insert themselves into ...

... optimized Cas9 variants have been created in an effort to increase translation in a particular plant ... the development of disease-resistant crops to increase agricultural yield (Wang et al., 2016; Wang et... the intervening region into the plant genome. Created with BioRender.com. In recent years, deaminase-mediated...Addgene carries several useful CRISPR toolkits for creating plant expression plasmids, including plant CRISPR...