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...create a toxic product. The E. coli strain Most E. coli strains can be used to propagate plasmids, but...plasmids from the same group should not be co-transformed, so if you need two plasmids for an experiment...groups. Note the A -C compatibility grouping is an arbitrary designation, and plasmids from the same incompatibility...incompatibility group should not be co-transformed. +Actual copy number varies. See below for additional...stocks, agar stabs, or liquid cultures. Freshly transformed cells also give a higher plasmid yield that colonies...

...company.  Ultragenyx’s goal is to develop novel products for the treatment of rare and ultra-rare diseases...deposited the ClonTracer Barcoding Library, a library that “enables the high-resolution tracking of more than...Dimension Therapeutics, which was acquired by Ultragenyx Pharmaceutical in 2017. As one of the original...the targets that have the biggest potential to transform patients' lives. Previously Stegmeier was the ...

...Wanisch  Listen to the optogenetics podcast segment LOVTRAP is an optogenetic system that was recently developed...modifying EB1. EB1 normally sits on microtubules to attract other proteins involved in their growth and extension...promoting domain. Each half was fused with one of the LOVTRAP domains. When both halves of the modified EB1 protein...separation of its synergistic domains. This paper demonstrated that the migration of a cancer cell line equipped...fluorescent proteins (FP) with improved tissue penetrance and live cell imaging capabilities. Upon developing...Quantification of editing efficiencies simply involves tracking the ratio of mCherry-positive to eGFP positive...

...investigator. In basic science (as opposed to translational, biomedical, or applied sciences), people conduct...don’t tell us everything there is to know is a strange and exciting revelation. Science in any field can...postdoc) Getting more scientific and professional training after earning a doctorate Considered a "stepping...1-2 years) Often a gap-year position taken to transition to the next career stage (ex: grad school, med...duties ex: order materials, lab maintenance Administrative assistant: assists PI with admin duties Staff...reach out to the PI to get involved. It not only demonstrates initiative and drive, but also makes for an ...that you have new interests and that you want to travel in new directions. During my grad school interviews...

...large inserts can make lentivirus production frustrating - a researcher can complete an entire lentiviral...editing experiments. We aim to alleviate this frustration by producing, titering, testing, and providing...troubleshooting reagent production, because that's a distraction from your primary goal. Getting these quality-controlled...ready-to-use lentivirus from Addgene, you’ll also receive transfer plasmid DNA that you can use along with lentiviral...Addgene Blog Get the Most from Your Lentiviral Transdution Using Virus in Your Research: A Primer for Beginners...Beginners 5 Tips for Troubleshooting Viral Transductions Additional Resources on Addgene.org Visit the...

...across the 11 candidate transcript targets analyzed. CRISPR-Csm presents an attractive alternative for nuclear-RNA...off-target costs. Additionally, this system also demonstrated the ability to be used as an endogenous RNA ...! Colognori D, Trinidad M, Doudna JA. Precise transcript targeting by CRISPR-Csm complexes. Nat Biotechnol...delivering them to a tumor - they used focused ultrasound (FUS) to heat the tumor area, as the image below...activity of CAR-T cells within tumours via focused ultrasound. Nat Biomed Eng. 2021 Nov;5(11):1336-1347. doi...

...responses are varied; one outcome is directed transcription from a given promoter. To harness the power ...the RR (OmpR) via phosphorylation, promoting transcription (see figure below). Subsequent exposure to red...response regulator via phosphorylation, promoting transcription of a GFP reporter. Subsequent exposure to red...system less bulky and easier to use with other strains of E. coli, Schmidl et al. condensed the system...types of proteins, including Cas9 and dCas9-transcription factors, for which low expression levels are... optimal. These tools are compatible with many strains of E. coli, as well as with other plasmid-based...

...similar for both, though tissue samples do require an extra homogenization step. For this blog, we're going ...but bone or especially fibrous tissues may need extra steps.  Fragmenting  The next step is break the ...your protein of interest is a low abundance transcription factor or cofactor. In such cases, the gentler...Proteinase K to purify the DNA and then determine concentration.  Protein Capture After crosslinking and sonication... purification kit or with a phenol-chloroform extraction.    Fig. 1: Schematic overview of ChIP..., it may be a good idea to add "patience and an extra-methodical approach" to the top of your protocols...

...not entirely. What truly unites us all is the frustration of an experiment failing because of a small, ..., avoidable mistake. Or worse, when you're trapped in a pattern of small errors that significantly impacts...rushing, no matter how hard I tried to build in extra time before the experiment. What fixed it was not...pipette tips to match your plate layout can help you track samples as you add them. Moving your reagents from...the headphones aren’t actually playing music. Nontraditional cues are an option, too - one of my coworkers...and open to new methods, you’ll be able to find strategies and systems that set you up for success in the...

...disease. CRISPR has made genomic SDM relatively straightforward, but plasmid-based SDM has lagged behind. While... and the nonmethylated PCR products are then transformed into E. coli to undergo nick repair. This method...homologous recombination. In most recombineering strategies, only 3-4 phage proteins are needed and these...containing the desired mutations, to amplify both strands of a plasmid template. In addition to containing...template plasmid, these PCR products are directly transformed into bacteria expressing recombineering proteins...Sci Rep 6:19121. PubMed PMID: 26750263. PubMed Central PMCID: PMC4707547. Additional Resources on the ...

...community. As a result, I have occasionally pondered translating my research into a startup company. However, ...school and turn it into a biotech company. Transitioning an idea from academia to a startup Mike Koeris... biotech startup, Mike explained, his career transitioned away from the bench and became all about finding...their idea at various levels of complexity and abstraction and convince people with diverse backgrounds ...company may begin to prove the technology. This transition takes around 1 – 2 years. The product is the...into it.” Have any other helpful tips to help transition a project from academia to a biotech startup ...

... a lack of transparency in most review processes. Preprints and their feedback, in contrast, are immediately..., and increase their impact. Open access and transparency Another major benefit of preprints is their ...record of changes, critiques or endorsements. In contrast, journal-based peer review is usually a confidential...committees encourage and consider preprints as demonstration of productivity. Part of an open scientific ...Preprints can be one tool in a combination of strategies for your scientific communication needs. Going...

...a variety of assay types. Be sure to check the spectral properties of the fluorophores you are considering..., each other, and your target. I love FPbase’s Spectra Viewer tool for comparing different fluorophores... (HRP) and Alkaline Phosphatase (AP). Various substrates for each of these enzymes allow you to detect...Endogenous carbohydrates added to antibodies post-translationally. 4) Modifications made to antibodies, such ...remove the incompatible agents by dialysis or gel filtration.    There is, of course, a lot more to explore...Stephenson W, et al (2017) Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14:...

... this transient construct retains functional Type IIS sites and will be re-digested. In contrast, formation...into a modular cloning strategy. Sometimes referred to as MoClo, this strategy uses the Type IIS restriction...Type IIS restriction enzymes are unique from "traditional" restriction enzymes in that they cleave outside...containing plasmid such as pX330. This cloning strategy not only makes it easy to create a single gRNA-expressing...

...localization in cells, or even single-particle tracking of the bound complex. There are so many FPs available...fragments are named according to the number of strands in the canonical beta-barrel structure. Most frequently...frequently, the proteins are split between strand 10 and 11 to yield large (1–10) and small (11) fragments...in N-terminal fragment Constant rate of p53 tetramerization in response to DNA damage controls the p53 ...TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells... can even be bright enough for single-molecule tracking in live cells.  Controls  When designing experiments...

...blot Run your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize...from having to run a whole new gel and membrane transfer. Let’s get started! Before you begin In the best...antibody binding to antigens. Ingredient Concentration or % Glycine or Glycine-HCl 25–200 mM ...more of your target protein. Ingredient Concentration or % Purpose Tris HCl 62.5 mM Buffer...whether that involves adding fresh chemiluminescent substrate or imaging your fluorescent probe. Then, if you...

...approved drugs so that your results are viable for translation to human patients. Picking receptors The receptor...nonnative ligands. However, they retain their intracellular interactions and still set off the same pathways... target effects. These synthetic agonists can penetrate the brain and some, such as varenicline, are already...Varenicline has been further modified to create ultrapotent PSEMS (uPSEMs) that have drastically increased... A, Basu J, Michaelides M, Sternson S (2019) Ultrapotent chemogenetics for research and potential clinical... specifically designed Gi-coupled receptor in transgenic mice. Nature Biotechnology 17:165–169 . https...

...China’s Ribobio, the illustrious speaker list attracted a host of sponsors based all over the world. With... market where one can shop for components of traditional Chinese medicine. There were hundreds of stalls...'ve overlooked regulatory fragments of these transcripts. I remember when small RNA was just something...’t give tRNAs much thought. High throughput transcriptome sequencing methods including work done by the...knew…I had barely heard of these). These single stranded, covalently closed RNAs seem to be conserved in...Using the Tet-on system, his lab has created a tetracycline cas9 inducible knock-out system. His lab is ...

...interactions (e.g., scores >80) are transformed to 100. This transformation (illustrated in the graph below) is performed...design tool Crispor by Max Haeussler. With a few extra lines of code, we enabled it to return guides and...the DNA target. CFD scores are then modified (“transformed”) such that weak interactions (e.g., scores <...re-create a match for the active guide. With these constraints in mind, we implemented the following algorithm...

...blastn), Standard Protein BLAST (blastp), and Translated BLAST (blastx). NCBI has a terrific getting-started...sequences (blastn)” program for blastn so that you can extract practically every single reliable base pair from...longer alignments to compare with your sequencing trace file. Unlike megablast, the regular blastn program...gaps in the nucleotide sequence, you can run a Translated BLAST (blastx) search to check for expected ORFs..., due to the additional searches involved in translating the nucleotide sequence into all six possible...choose for your sequencing result and can easily translate it, we recommend using blastp over blastx. The...