...designed to shuttle between species. Figure 1: Shuttle vectors are vectors that can function in ...are used across species: replication, selection, and expression. Replication It’s usually not enough to...chromosome has a single origin of replication (ori), where DNA replication begins, so a bacterial plasmid...Eukaryotes are a bit more complicated. Yeast vectors may contain an autonomously replicating sequence (ARS), a...fast-growing populations of cells, the vector needs to replicate alongside its host, or it will be lost from the...compatible ori that will be recognized by its host’s replication machinery. A shuttle vector designed for unrelated...experiment. Yeast shuttle vectors have these replication sequences for their yeast targets as well as ...

...Figure 1: Flowchart showing the process for using pooled lentiviral libraries. A) Amplification, validation...you.  Starting from plasmids requires amplification, verification, and lentiviral production steps before...the same proportion after amplification as they were prior to amplification. And as anyone who has worked...pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to grow on ...into HEK cells allows production of lentivirus. Purification of this lentivirus results in the pooled library...//pubmed.ncbi.nlm.nih.gov/27366891/   Amplification When you request a DNA pooled library from Addgene...might receive a similarly-sized aliquot from an amplification done by Addgene.) This small aliquot is not ...

...plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems” require...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...selection markers. Restriction modification systems Because restriction modification systems consist of two enzymes...used as biological tools for a wide range of applications in molecular biology or in vivo studies. Let...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin...CcdA (antitoxin) where CcdB inferes with DNA replication by binding the GyrA subunit of DNA gyrase and...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...

...successful and meaningful career in the grants profession: 1. Build on your existing skills & passion  Skills such...development, certification, degrees, foundations, coaching and even Twitter Chats dedicated to the Grant...the candidate application process, the candidate’s ability to write a grant application through a written... much more than writing. In fact, many grant applications and grant makers have moved away from long written...Now they require grant seekers to use online applications with significant character limits for the written...written portion of the grant application. They have increased their demand for stronger budgets with clear...duties can range from coordinating the grant application and submitting it, to evaluating the capacity...

...can make this fun activity relate to STEM.” Figure 1 shows the activities we had for my school’s Math &...my opinion, it was more important to communicate the applications of CRISPR over the details of how it...m a father of three, a scientist and science communicator, and I strive to be a volunteer and contribute...recent experiences. STEM in elementary schools - catching them early I’m fortunate to live in a town with...unlike elementary schools, middle schools have dedicated science teachers. I tried contacting our school...simplistic and I would insult the students. Too complicated and I would quickly lose their interest. I won...great feedback from teachers and other science communicators.  What can CRISPR do? I spent more time on...

...two (Knutson et al., 2010a,b).      Fig. 1: Undergraduates in Provost’s CURE prepare to analyze... put it best when he stated, “the value of an education . . . is not the learning of many facts but the...possible to bring the kinds of transformative educational experiences afforded to those fortunate enough...which can lead to greater success in meeting educational goals (Kowalski et al., 2016; Scott, 2021). In...CUREs are beginning to transform undergraduate education in the sciences by making a greater number of ...scientific research more inclusive. CBE Life Sciences Education. 13(4):602-606. Brian, D. 1996, Einstein: A Life...positive STEM attitudes in CUREs. CBE Life Sciences Education. 21(4):ar74. Duboue, E., Kowalko, J., Keene, A...

... such a popular model organism? Zebrafish (Figure 1) were initially recognized as a useful vertebrate ... are not ideal for every application. Disadvantages include their duplicated (i.e., redundant) genome,...mutant, which regulates a developmental pathway implicated in congenital heart defects and cancer. These...DNA into the genome is useful for a variety of applications. For example, specific tissues such as blood...pathways control this process. Additional zebrafish applications and conclusions Zebrafish are also popular outside...—have proven useful in a variety of research applications. Lineage tracing studies highlight how these...Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." Methods...

...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...Addgene has a growing catalogue of viral preps available to request. In this post, we’ll walk you through...minimized by using an endotoxin-free plasmid purification protocol. To minimize the immunogenic properties...posted on the material page for the corresponding catalog item (see maps section for images).     ...Lentivirus Production Lentiviral preparations ("-LV” catalog items) are produced in 293T cells using the 2nd.... Concentrated lentiviral preparations (“-LVC” catalog items) are generated from lentiviral preparations...typically prepared using endotoxin-free plasmid purification kits.     Figure 4: Schematic of the...

...antibody bonding strengths.    . Steps of an IP: 1) Protein extracts from cells or tissues contain a ...a protein’s stability or post-translational modifications, or study how a target protein interacts with...Protein A and Protein G are not suitable for all applications, such as protein isolation from serum. In this...capture antibody interaction and/or the downstream application. Typically, if the target protein will be detected...and an isotype control antibody. Alternative applications Oftentimes, an IP-appropriate antibody simply...identify DNA binding proteins involved in histone modification. Similarly, RNA IP (RIP) can be used to isolate...resources References:  Björck L, Kronvall G (1984) Purification and some properties of streptococcal protein...

... reached labs in over 75 countries. Figure 1: SARS-CoV-2 proteins and other components. Image from...a method that uses loop-mediated isothermal amplification after reverse transcription (RT-LAMP) and a ...receptor for SARS-CoV. The lab also found that cathepsins play a role in SARS-CoV infection and deposited...deposited plasmids for the expression of cathepsin S, L, and B. This became a great starting point for SARS-CoV..., Zengel JR, Sathyan V, Nguyenla X, Van Dis E, Catamura C, Yamashiro LH,  Begeman A, Stark JC, Shon DJ...

...template including the desired edit. Figure 1: Prime Editing schematic. Created with BioRender.com...BioRender.com. The pegRNA specifies both the genomic location to be edited and the sequence of the edit, so ...its design changes for every new prime editing application. And optimizing its sequence is important, as...left), while pegRNAs that use other bases in this location bind to Cas9 with canonical sgRNA structure (right...structured motifs like mpknot, use the pegRNA Linker Identification Tool (pegLIT) to create linkers that minimize...

... end sequences (R and L ends).  Figure 1: (A) Components involved in each type of CRISPR-Transposon... and have been adapted for a wide variety of applications - as mutagenesis tools for genetic studies (... The process also creates a 5-bp target-site duplication (TSD), a hallmark characteristic of the Tn7-family...the new streamlined VchINT system was shown to catalyze insertion of mini-transposons up to 10 kb in size...at one or multiple desired genomic loci. Many applications of these systems have already been demonstrated...

...blood test for HMGB1, the high mobility group box 1 protein. Normally localized to the nucleus, if the...cancer. These later stages of a cancer diagnosis indicate, for Stage 3, that the cancer is large in size...size and may have spread while Stage 4 indicates that the cancer has spread from where it originated to ... dire prognosis for mesothelioma patients is to catch the disease earlier. New techniques are currently... working to fight the cancer.  Beyond early modifications to standard treatments and early detection, ...

...testing. You can find Nick on LinkedIn. References 1. Skloot, Rebecca, and Bahni Turpin. The immortal life...Marker-independent identification of glioma-initiating cells. The International Cell Line Authentication Committee...propagation of the line, and can include assays for authentication and contamination) and cell lines can become...let alone the human cost. Here are just a few publications which have since been retracted due to cell ...

...mammalian expression from human elongation factor 1 alpha Constitutive  Tends to give consistent expression...resistance gene and the origin of replication are necessary to replicate plasmids and make sure the cells...length version is strong with high expression. Truncated promoters are constitutive with lower expression...metabolic operon Inducible by arabinose and repressed catabolite repression in the presence of glucose or by competitive...

...antibiotic selection. Essentially, this gene is a 2-in-1 providing both bacterial and eukaryotic selection,...common antibiotic resistance genes and which applications are best (or worst) for each antibiotic. The...is expensive (relative to amp), and for some applications, the benefit it offers is marginal. Fortunately...antibiotic resistance ultimately comes down to your applications and needs. The more niche and specific your ...

...such as model systems and primary cells. References 1. Doench, John G., et al. "Rational design of highly...is only effective when sgRNAs target a specific location on the gene. For CRISPRa, sgRNAs were targeted...Calabrese pooled library and lentiviral prep here! Applications for the Root and Doench CRISPR libraries Overall...et al. "A protein-tagging system for signal amplification in gene expression and fluorescence imaging....

...fun together! Laura’s Top 10 Event Planning Tips! 1. Take suggestions Some of Addgene’s best events have...be an obvious one, but don’t make things too complicated for yourself (especially if you are the sole ...sole event planner)! If you have a very intricate itinerary, there is a higher likelihood that something ...Delegate- it will save you time and sanity!    10. Communicate Make sure people know that there is something...

...drives our bioinformatics needs       Fig. 1: QC'ing data at Addgene is like standing under a Niagara... from depositing laboratories and associated publications, but our quality control (QC) standards have...such as 96-well microplates, and thanks to our dedicated product and development teams, frequent updates...can be easily shared with others without any complicated setup. We created, tested, and debugged each ...

... of low-toxicity RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using...experimental design and its potential for clinical applications. Specifically, efficacy rates of Cas7-11 were...place, while increasing the number of potential applications. But they weren’t done engineering quite yet... at the bench and potentially in therapeutic applications. The potential of CRISPR has yet to fully taken...