...definitely help in understanding if your strategy will be successful, and to avoid easy mistakes that could affect... and the expected result is due to a PCR mistake or a mistake by the DNA analysis software. Now that you've...hopefully obtain your coveted plasmid with no substantial delays. Design, design, design Before diving ...many softwares out there than can help you at this stage and that can be used to simulate in silico cloning... of insert compared to the backbone plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert...antibiotic in the plate correspond to the antibiotic resistance marker present in your plasmid. You should also... mix in different bacterial strains (stbl2, NEB stable, etc.), and/or use a different temperature (30°...

...that is resistant to the toxic effects of CcdB. Another commercially available, CcdB-resistant strain ...components that are key to the cloning reaction. Understanding the specific components is essential for choosing...actually contain your insert of interest. Simply stated, ccdB makes cloning easier by selecting against...But exactly how does ccdB accomplish this? Let’s start with a brief history of the gene and how molecular...non-recombinant vectors (re-ligated empty vectors, for instance) would still express ccdB and therefore would ... investigator’s gene of interest.   CcdB resistant E. coli strains complete the system But ccdB alone...contains the F plasmid (F’ strains) will also be resistant to CcdB, as the native ccdA will be present. Most...

...Communication blog series, you’ll hear from Susan Keown, a staff writer at the non-profit Fred Hutchinson Cancer ...Center. Susan Keown’s science communication journey started with something unexpected: termites. “I was really...says Susan. Her passion for insects lead her to start a PhD in entomology at the University of Maryland...’m like, “Oh my god, what can I do?! This is a mistake! Why did I even come [to grad school]!” He asked...know if she liked science writing. She decided to start a blog to test it out and discovered she not only...continued working for Palladian Partners remotely, but started looking for a new job because “it kinda sucked ...hired a full-time social media manager. “I usually start out my days looking through our Twitter feeds and...

... at MIT) where he worked on understanding bacterial antibiotic resistance. During this time, Dr. Koeris...In the third installment in our podcast series, we chat with new Addgene Board Member, Michael Koeris...this idea, Drs. Koeris and Lu founded the biotech startup company, Novophage. As you'll hear Dr. Koeris explain... decided to develop phage for use in a biotech startup 12:16 - 15:15 : Why Sample 6 began to develop tests...Koeris to personally take on the risk of founding a startup company? 32:20 - 34:37 : Whose responsibility is...Sample 6 website Check out Dr. Koeris' Website, biotechstart.org Read Sample 6's Paper on Bacteriophage-based...Other Posts in Our Podcast Series Get Additional Startup Advice Learn How to Be A Good Manager from Addgene...

...present. Using a Standard Curve To do that calculation, you’ll need to include a standard curve in your ...your standard curve to plot to a linear equation (remember y = mx+b?). Figure 5: A standard curve...that relies heavily on antibodies is ELISA, which stands for Enzyme-linked Immunosorbent Assay. ELISA are...readers can detect is 4.0, so if you max out your standard curve and it reaches an asymptote, you won’t be...wells in that range of optical densities. If your standard curve does not have a linear portion or the OD...when you repeat the experiment by diluting your standard curve, performing a serial dilution of your experimental...Once you’ve identified the linear portion of your standard curve, you can calculate the concentration of ...

...designs and has improved protein yields and stability. This stabilized spike protein can be used for the development...LH,  Begeman A, Stark JC, Shon DJ, Fox DM, Puschnik AS, Bertozzi CR, Carette JE, Stanley SA, Harris E, ...Apr 30, 2020 and last updated Jul 12, 2021. What started out as a small collection of plasmids for coronavirus... of cathepsin S, L, and B. This became a great starting point for SARS-CoV-2 researchers to investigate...Calabrese (activation), from Addgene to better understand which human genes are important for SARS-CoV-... packaging) and contain an IRES-Puro marker for stable cell clone selection. For more information on these...JS (2020) Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science 369:1501–1505. https...

...existed! Addgene recently started automating a few protocols to make standard lab processes easier and ...compared to a lab assisstant filling about 200 vials in 45 minutes by hand. How can you start automating your...robot and to find out how you can get one. As a starting point, I would recommend conferences like Lab ...majority of Addgene plasmids are shipped as agar stabs in glass vials. Have you ever wondered who fills...fills all those vials? Well, it used to be a lab assistant, but with increased plasmid demand it was too ...can do to get into the robotics spirit include: Start using electronic multichannel and single channel...Addgene would love to move forward and automate. For instance, vial labeling would be much easier with a Tube...

...9514755. Richard Gronostajski: NFI-related diseases of neural development Richard Gronostajski is a professor...Paula Videira, Michael McMurray, and Richard Gronostajski who each are being awarded free plasmids form...disorders of glycosylation Paula Videira is an Assistant Professor at the Faculty of Sciences of NOVA University...Applied Molecular Biosciences (UCIBIO) focused on understanding the physiological and pathological roles of ...biosynthetic pathway and have a wide range of clinical manifestations. PMM2-CDG is the most frequent CDG type characterized...University of Buffalo. His research focuses on understanding the structural determinants of protein-DNA interactions...they affect brain and muscle development. The Gronostajski lab has generated mice wherein it's possible...

...deconstruct and understand this complexity using existing C. elegans mutants. For instance, we use a mutant...research on worm locomotion, but our worms are constantly moving so we need good ways to monitor and track... C. elegans molecular biology are based on the standard worm genome. While C. elegans are often thought...and so far most of their funding has come from kickstarter and fundrazr projects). This allows the project... to a wider group of people. This is a perfect starter project for our computer scientists, many of whom...interested in biology but don’t really know how to start applying their skills to it. The Kittybiome project...Funding Makerspaces One of the main challenges when starting a new project at a makerspace is finding the financial...

...number is then determined by comparing results to a standard curve. For CNV assays, it is recommended to use... (Ma, 2014.) As with all qPCR assays, samples, standard curves, and normal controls must be run in triplicate... the variance seen within multiple runs of the standard curve. Signaling molecules: Sybr Green and Taqman...noise, due to the large amount of dsDNA in most starting materials, and is not particularly sensitive. ...each reaction, math happens (this time, Poisson statistics), and the readout gives an absolute value of ...concentration values, rather than by comparison to a standard curve, and most results can be confidently interpreted...Bell, 2018.) Even though it does not require a standard curve or a normal control, it can take a few hours...

...plants, and agricultural crops in particular, resistant to disease. They also devise biosensors designed...that they work well in plants and give similar stability to transcripts as the ones that people use all...all the basic and necessary elements in order to start using the MoClo system. The paper also gives quantitative...have any particular tips for those looking to get started with MoClo? Patron: The parts will be familiar ...the parts that you need from the kit and getting started making your own constructs with Golden Gate MoClo...conceptual barrier. Sometimes people don’t know how to start. They might want to use our promoters and terminators...they don’t have any prejudice about it. They just start and go ahead. I can see science speeding up. Addgene...

...verify the starting methionine of a gene but the blastx results start the alignment at a more distal amino ... available, we primarily use Standard Nucleotide BLAST (blastn), Standard Protein BLAST (blastp), and ...Translated BLAST (blastx). NCBI has a terrific getting-started guide for BLAST, which includes a simple explanation...differences below. Optimizing blastn searches On the Standard Nucleotide BLAST page, the first decision to make...homologous regions, which by default are ignored in a standard blastx search. An alignment omitting repeated ...the sequencing result, we often choose between a Standard Protein BLAST (blastp) and blastx search to verify...sequence alignments that BLAST is unable to handle, Clustal is our frequent choice for pairwise or multiple...

..., such as the starting material (DNA-based vector or purified RNA) and the initial state of the dsRNA ...experiment functions as the building block of our understanding of complex biological processes. Many tools ... knockouts have long been considered the “gold-standard” for LOF studies, the high gene copy number found...function due to the use of exon skipping, alternative start codons, and/or the phenomenon of transcriptional...transcriptional adaptation (Smits et al., 2019; Sztal and Stainier, 2020). Additionally, a study that directly compared...approaches and considerations present at the first stage of an RNAi-based investigation. Creation of the ...gene silencing efficacy, thereby requiring a substantial time investment. Once the target mRNA is cleaved...

...knockouts.   Star Trek: Into Darkness (2013) which is the remake of the space-melodrama Star Trek II: The...into the twenty amino acids. Once scientists established that DNA encoded all the information necessary...were exploring were space and robots. Warping to stars in space ships and the rise of killer machines that...Released in 1997, GATTACA is an American SciFi film starring Ethan Hawke, Uma Thurman and Jude Law. The movie...novel, Inferno (2013) and the movie version (2016) stars Tom Hanks as the protagonist (Langdon). The plot...engineering in the recent SciFi movies. Rampage (2018), starring Dwayne “The Rock” Johnson, calls out CRISPR as...pegRNA (prime editing RNA), the prime editor can install all possible point mutations, delete short stretches...

...special about her pure interest and curiosity to understand my work and me, especially as much of her focus...environment filled with people who are interested in understanding the work you do, in addition to being genuinely...bigger projects I was tasked with was creating and sustaining a biweekly report of our social media analytics...Addgene before. This meant I was responsible for establishing a new report and routine across the marketing...excited but overwhelmed; I had no idea where to start. Thankfully, my managers set aside time for me to...template to build out a report on. I was able to understand what metrics would be useful and how to organize...with the larger content and marketing team, and I started preparing for my first official professional presentation...

...work gets done Managers do this by removing obstacles that stand in the way of the doers and by ensuring ...just have to know it when you see it? “The Three Stages of Your Career: Doer, Manager, Leader” by Charlotte...importance of these very different roles. At any stage in our careers we may be in a cycle of doing, managing...are the ones who do the work Doers focus on understanding what is expected of them and learning new skills...look to the future Leaders must get new things started and then step back to let others take the reins...productive culture and to correct systemic problems. Understanding these three roles allows you to identify what...the requirements of the current task. Most of us start as doers, some become managers and some take on ...

...single cell isolation and downstream sequencing (Standage-Beier et al. 2019). TREE is a real-time, fluorescent...in emission spectra from BFP to GFP. Image from Standage-Beier et al., 2019. The team put TREE to the...reporter proteins. Protein translation requires the start codon of AUG immediately downstream of a kozak sequence...mScarlet reporter, luciferase reporter, and neomycin resistance reporter. Image adapted from Katti et al., 2020...generated a silent GFP construct, that contains an ACG start codon mutation, and integrated it into human and...As the initiation of protein translation at the start codon ATG is universal, the Dow lab successfully... including mScarlet-I, luciferase, antibiotic resistance markers, and other enzymes such as Cre-recombinase...

... laboratory tool often employed to establish cell lines that stably express a gene of interest. While ...background of the lines you are using, it is best to start from scratch. Obtain a vial of the desired cell ...production and infection.     Great transduction start with great virus If having happy cells is the first...extreme changes in osmotic pressure and pH can destabilize the viral envelope. One drawback of the multiple...The effect of freeze-thaw cycles on lentiviral stability depends on a variety of factors such as the transgene...al. found that lentivirus produced at pH 6 are resistant to multiple freeze thaw cycles. Consequently, ...prep!) and infect. While this approach is a great start to a successful infection, one point to consider...

...new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the sample ...acquired all fluorophores throughout the sample are constantly exposed to light, not just those in the focal...can a a high enough density of red-photons be establish for this to happen.Using red light has a second...within the depth of field of the objective (the distance in the axial dimension that will appear in focus... effects of high light doses to the cells. Thin static samples Ex: Fixed monolayers of cells or thin (... requirement for high temporal imaging speeds. Standard fluorescence widefield microscopy can be combined...require both optical sectioning, imaging over large distances and high temporal resolution. Traditionally, this...

...conclusion. Advice for Telling a Good Story Start at the start Any good story needs a solid point to kick...at. Whatever you’re working on, start wherever it makes sense to start and try to be as chronological ...almost all of us are programmed to appreciate and understand. We build narratives around the self, too — ...author. When I raised this, he was pretty angry and started arguing with me. First I calmed the situation down...for all of us.” This is a classic example of the STAR (Situation, Task, Action, Result) interview technique...and engage with and what parts you can leave out. Start viewing your work in the form of a story, and you...telling it. End at the end A story needs a good start point and it needs a good end point. One thematically...