...screening blog post for more information.   Figure 1: Result of a blue-white screen. Image from Stefan ...Because Sanger sequencing can usually sequence just 1 kb of DNA, custom primers are especially helpful in... of the entire plasmid. Please note that NGS verification is more time-intensive than Sanger sequencing...

...additional flexibility in pseudotyping.   Figure 1: Genome of a prototypical gamma-retrovirus.    ...an SV40 promoter, and Neomycin selection.   Table 1: Gamma-retrovirus glossary Plasmid Type Element... be used interchangeably. Are γ-retroviruses replication incompetent? Yes, as with lentiviruses, the genes...

...Last updated Sept 1, 2020. This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill...the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There is consequently...Giovannangeli C, Jasin M, Brunet E (2014) Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous...

...introduced, they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...more about them on our CRISPRa blog! Fig. 1 CRISPR activator and repressor mechanisms Want...interference (CRISPRi) targets a catalytically dead Cas9 fused to a transcriptional repressor to your ...

...are working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...constructs. His work was acknowledged in hundreds of publications including Cell, Nature, and other journals. ...

...main differences between Cas7-11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7... targeting single stranded RNA.   Table 1: Fast facts on Cas family proteins and genome editing...a defined site to form a clean DNA break, Cas3 locates its target sequence and initiates single stranded... out large regions of DNA and has anti-viral application potential as a result. Fast facts – PAM requirement... has been proposed to be useful for the same applications (RNA knockdown, RNA editing, etc.). The main...

...the lab running like a well-oiled machine. Lab tip #1: Glove trick Putting on lab gloves can sometimes be...With barcode tracking, you will always be able to locate materials quickly and easily. Learn more about ...Additional Resources on the Addgene Blog New Educational Resource: The Addgene Videos Page 5 Science Rap...

...company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking...to take notes after each interview.   Tailor your 1-hour presentation to your audience Start by asking...accomplishments and demonstrate your science communication skills. Know your audience. Connect your prior...codes for interviews. You can also ask HR for clarification on the dress code for the interview. Greet each...

...writing and hopefully increase your success rate. 1. Submit your grant early! First off, and perhaps the...involved so it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback...Series 4. Become a science communicator More and more fellowship applications are asking for a 200-300 ...good quality application. Even following this advice, only about 50% of my grant applications have been ...do need to be able to research and write your application, then give it to someone else to read and / or...it). It’s easy for reviewers to spot when an application is rushed (whether the idea isn’t properly researched...researched / developed or the application is full of typos) and these are unlikely to get past the initial...

...self-assembly for applications in health, energy, and sustainability.   References 1. Pardee, Keith, et...detects samples at clinically relevant concentrations (1-3 fM; 1x106-fold lower than before), and our combined... journal Cell just six weeks later. The Zika publication timeline I think we can speak for all the authors...research, but the urgent need for the test did catalyze our resolve to get the work done as quickly as... dozen versions of the toehold switches and amplification primers. This helped make sure that we had a...this includes primer directed isothermal RNA amplification, new toehold switch designs, CRISPR/Cas9-mediated...our electronic reader that provides portable quantification of diagnostic results.  Our approach now detects...

...: it’s dCas9… with no modifications). Ready to get started?   Figure 1: Break free from the ball...?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks from parental to... when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9....for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable, otherwise...vivo using dCas9–peptide repeat and scFv–TET1 catalytic domain fusions. Nat Biotechnol 34, 1060–1065. ...

...Emerging Investigators Podcast breakdown Intro Chapter 1 - Introduction to Mark Springel, Olivia Ho-Shing, ...Additional Resources from the Addgene Blog Find education resources Use our Plasmids 101 eBook to teach ...

...Fragment? Consider the classic IgG antibody (Figure 1): it is composed of four peptide chains (two identical...that actually bind antigens.      Figure 1: IgG antibody structure with important features labeled...fragments described above can be used for similar applications and have similar advantages, though with some...

...Glycine-HCl 25–200 mM SDS 0.1%–1% (w/v) Tween 20 (optional) 1% (v/v) HCl Adjust to pH 2.0... tool to identify a protein of interest in a complicated solution like cell lysate. But they can be a ...

... Joachim Goedhart Podcast breakdown Intro Chapter 1 - How Joachim ended up at the University of Amsterdam...Twitter and has helped us update many of our educational resources. Listen to hear all about Professor...speeds science. Chapter 8 - The importance of communicating research findings through social media and other...

...forms of science communication. Click here to listen to the interview with Tom McFadden!   1. Rosalind Franklin... from Addgene.      Check out more science education tools like Verse Two and Fill in the Planck at...with us. Tom McFadden is a YouTuber and science educator looking for creative ways to implement the next...next generation science standards and communicate science. Learn more about Tom on his youtube channel ...Community Check out All of Our Podcasts Find Great Educational Content in our Plasmids 101 Series Additional...

...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are ... enzymology of DNA repair enzymes.     References 1. Shuman, Stewart, and Christopher D. Lima. "The polynucleotide...thermostable DNA ligase-encoding gene." Gene 109.1 (1991): 1-11. PubMed PMID: 1756968. 7. Lauer, G., et al. "Cloning...or DNA base pair mismatches (8-11). For some applications, there cannot be any end-joining activity, and...regions. Why use a high fidelity ligase? Numerous applications have been developed to take advantage of the...Taq and other thermostable DNA ligases. These applications detect specific nucleotide sequences with high...rounds of melting and annealing allows linear amplification of the probe ligation product. With the right...

...CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative numbers of CRISPR plasmid ...new CRISPR depositors each year.    Figure 1: Addgene CRISPR distribution and deposits. (A) Cumulative...estimated scatterplot smoothing. (C) Number of requests per year for pooled libraries, categorized by function...insertion via a chemical modifier. We used these categories to see what kinds of CRISPR tools people were... pooled libraries, although we used different categories (Figure 2C). Note that in Figure 2, the numbers...Cumulative number of deposited CRISPR items, categorized by CRISPR function, by year. (B) Number of requests... years.  Libraries are divided into different categories based on function: knockout (KO), activate, inhibit...

...interrogated by the optical system (Figure 1).   Figure 1: Cells expressing a particular surface ...populations of cells in a complex tissue or sample (1). When combined with cell sorters, this technology... it passes through and scatters in different directions. The degree of scatter is captured by detectors...Light that scatters sideways to the path that it was initially traveling is called side scatter (SSC) and...granulocytes in a plot of forward scatter (FSC) versus side scatter (SSC). Users can gate around their...protein composition. It is used for a variety of applications such as measuring target protein expression ...expression levels, assessing post-translational modifications, determining cell health, analyzing cell cycle stages...

...principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes... dopamine D2/D3 receptors. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-78827-9 Breen... Biophysica Acta - Biomembranes (Vol. 1838, Issue 1 PARTA, pp. 15–33). https://doi.org/10.1016/j.bbamem...emission wavelengths, which are pivotal in their application in biotechnology (Grimm & Lavis, 2022). These... substrates/peptides and antibodies. Example applications of this include coupling to an antibody, whereby...whereby excitation of blue light will indicate where FITC is bound through green light emission. Key considerations...typically bind to. Stability of fluorescence: For applications that involve ranges of pH, and significant lengths...