...Neveu, 2015). Origin and purpose of MXS chaining MXS-chaining was designed to create plasmids for fluorescence... principle behind MXS-chaining is repeatable, restriction-enzyme based (chaining-based) cloning. The modules...promoters Gibson et al., 2009 MXS-Chaining, BioBricks, Bglbricks, etc. Chaining-based methods (restriction enzyme-based...similarly apply MXS chaining to your experimental needs. Let us know how you use MXS Chaining by emailing us....org. Table 3: Chaining Based Cloning Methods Method Restriction Enzymes Chaining Direction MXS-Chaining...useful application of high-throughput cloning. MXS Chaining is one such technique and has been used to produce...example, modular constructs generated with MXS-chaining were used to label subcellular structures in live...

... stocks and use of the original Cell line you purchased, keeping all experiments carried out on low passage...you to track a cell line’s age and monitor any changes. Your tracking system could be as simple as a piece... stocks to work from. The future Attitudes are changing but yet more is needed to convince people of some...Lacks. New York: Crown Publishers, 2010. 2. Gold, Michael. "A conspiracy of cells." State University of New...

...tools, producing lentivirus, can pose certain challenges. Whether choosing a system that is the best fit...will provide an overview of some of the common challenges associated with producing and using lentivirus...make these optimization experiments a bit more challenging. With these considerations in mind, when planning...viral components; according the manufacturer these changes result in yields up to 25x higher than other available...such as Cas9. See figure 1 for an example of how changing transfection ratios can affect transfection efficiency...

...testing and a lack of well-characterized genetic tools make it challenging to engineer a specific function...areas with our color-changing flower, but to do that we’ve had to navigate the challenges involved in plant...Additional work has also identified and characterized mechanisms and components of the circadian rhythm...excited to see how this exchange of genetic tools facilitates plant science. Challenge #3: Intellectual property...fibers. And because working with plants can be challenging, there are a lot of unexplored areas in plant...plant synthetic biology. Challenge #1: Public perception of synthetic biology One of the biggest difficulties...manner. At Revolution Bio, we are addressing this challenge directly by developing a plant biotechnology product...

...Shen, and Michael Z. Lin. "Optical control of cell signaling by single-chain photoswitchable kinases."... Figure 1: Photoswitchable MEK1 (psMEK1). Is it possible to create other photoswitchable kinases? The...in the Michael Davidson Collection Find Photoswitchable Fluorescent Proteins ... lab present a generic design for creating photoswitchable kinases and use MEK1 as a model kinase. In ... MEK1 activity by attaching two dimerizing photoswitchable Dronpa (dron= a ninja term for vanishing, and...site of a constitutively active MEK1. This photoswitchable MEK1 is called psMEK1. When exposed to 500 ...psMEK1 also worked for creating three other photoswitchable kinases: psMEK2, psRaf1, and psCDK5. MEK2 is...

...created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA uses...Madison lab also created an inducible SRIRACCHA system (iSRIRACCHA) where Cas9 expression is activated ...these reporters, editing typically results in a change in the fluorescent readout. In this blog we will...focus on two fluorescent CRISPR reporter methods, SRIRACCHA and GEMCherry2. We will cover single base-pair...editing CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for...genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct. Image from Wen et al., 2017...for easy screening of different gRNAs by simply changing the gRNA used when transfecting your cells.  A...

...  Quantitative polymerase chain reaction, qPCR Quantitative polymerase chain reaction (qPCR) also known...What is polymerase chain reaction? If you have ever worked in a molecular biology laboratory you have...have likely done a polymerase chain reaction (PCR). PCR is an in vitro method in which a small amount of...thus a heat resistant enzyme that can withstand changes in temperature. Taq was first identified in the...standard curve, which can be time consuming and challenging to get right. ddPCR uses a water-oil emulsion...Quantifying low abundance targets Monitoring subtle changes in target DNA levels Multiplex PCR Amplify... Resources on Addgene.org Protocol: Polymerase Chain Reaction (PCR) Protocol: How to Design a primer  ...

...Continued development of new allelic exchange vectors Allelic exchange is a great technique for the scarless...allele exchange in bacteria." Plasmid 51.3 (2004): 246-255. PubMed PMID: 15109831. Pyne, Michael E., et...modification. This technique is frequently called allelic exchange. Here, a mutated allele introduced from a conjugative...off-target mutations. Ultimately, successful allelic exchange requires two sequential homologous recombination...counterselection vector is the pDS132 sacB-based allelic exchange vector. This vector contains sacB, an chloramphenicol... colonies isolated are those where the allelic exchange vector has undergone single crossover and integrated...escape). The rhamnose induction system is well-characterized and quite tight (Giacalone et al., 2006). The...

...going virtual for Addgene’s first AAV Data Hub Challenge, now taking place Dec. 6, 2022! Share your AAV...

...that this basic concept of gRNA design is still challenging for researchers using CRISPR, and that standardizing...paraphrased in the tweet below. The complexity and challenges of homology directed repair (HDR) are echoed ...s a list of blog posts to help you tackle the challenges of DNA repair (with bonus gRNA design resources... DNA for Enhanced CRISPR Genome Editing Chris Richardson and members of Jacob Corn’s lab describe how ...a brand new CRISPOR manual to kick off 2018! "Mechanisms of precise genome editing using oligonucleotide... donors". Miura, Hiromi, Rolen M. Quadros, Channabasavaiah B. Gurumurthy, and Masato Ohtsuka. Nat Protoc...Creating knock-in mouse models with CRISPR has been challenging, with protocols using dsDNA templates achieving...

...Streak Plate Challenge, Westenberg’s course includes Winogradsky columns and characterizing the microbes...plate technique. Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs a redo :-( @SandTBioSci... the lab used three different species for the challenge, but one species always outcompeted the other ...it would be a perfect fit for the streak plate challenge. Chromoproteins are a subset of the fluorescent... distinguish colonies from one another in the challenge. To create a successful streak plate, first the...colonies in the last quadrant after incubation. The challenge is usually successful for the students. “They ...colonies. I should probably make it a bit more challenging, potentially by using a thicker bacterial suspension...

...thanks to Alex Chavez for taking the time to sit down and talk with us.  Alejandro (Alex) Chavez is a member...episode of the Addgene podcast, we sit down with Alex Chavez, a Clinical Fellow in Pathology at Masachusetts...Learn More About Alex's Work with Cas9 Activators Michael Koeris Explains His Path from Grad Student to Entrepeneur...

...Additional Career Posts on the Addgene Blog How did this change in labor laws come about? In March 2014, the White...Institutions are now preparing to comply with the changes to the law on Dec 1st, according to guidance issued...stipend levels. In theory, therefore, the salary changes expected for postdocs in many cases should be as...to gauge exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral...policies are one indicatator of how salaries may changes due to the FLSA. In 2014, 89% of institutions had...salary range. We have been able to gather data on changes to salary for some of the institutions that are...what they are doing, however, may face greater challenges. Current salary information that we have been...

...cold conditions. However, many of the detailed mechanisms that allow extremophiles to survive under these...fruit juice clarification, meat tendering, and oligosaccharide synthesis. A major incentive to use cold-active...folding at low temperatures, since mesophilic chaperones would not function well under suboptimal conditions...engineering a plasmid which contained cold tolerant chaperones (the Cpn 10/60 isolated from Oleispira antarctica...the figure below shows, E. coli expressing the chaperones protein resulted in higher levels of growth compared...discussions. Not only are such endeavors a welcome changeover from conventional academic settings, these are...

...I include in my chalk talk?" So what exactly is a chalk talk? On the surface, a chalk talk is quite simple...present a "chalk talk." However, few, if any, faculty job candidates have seen an actual chalk talk. Their...first exposure to a chalk talk is usually their own. This is a problem. The chalk talk is effectively ... hone a chalk talk. Yet, I often hear, "I have an interview next week and it includes a chalk talk. What...steps to chalk talk preparation: Define your research vision. Organize your vision into a chalk talk format...simple. It's you, a dry erase board, some markers (chalk is rare these days), and a room of 5-30 faculty ...format. Practice. Define your research vision A chalk talk could be your plans for your first NIH R01 ...

...variable light chain (VL) and variable heavy chain (VH) of an antibody. These two chains are connected ...developed hybridoma technology. But what about single chain fragment variables (scFvs)? scFvs weren’t developed... for additional rounds of panning and scFv characterization.    Figure 2: Phage display starts ...plasmid in bacteria (Ahmed et al., 2012). One challenge of bacterial expression is the proper formation...cytoplasm. expression in the presence of molecular chaperones that help scFvs fold properly in the cytoplasm...Lee T, Pope S, Riordan G, Whitlow M (1988) Single-chain antigen-binding proteins. Science 242:423–426 . ... of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proceedings...

...optogenetics.  Challenges of engineering channelrhodopsins There are three main challenges to engineering...machine-learning to overcome channelrhodopsin design challenges To overcome these challenges, the Arnold and Gradinaru...the expression of light-gated ion channels such as channelrhodopsins (ChRs) makes them light-responsive... shines on excitatory channelrhodopsin-expressing neurons, the channelrhodopsins respond by opening and...deliver channelrhodopsins to the brain. This delivery route concentrates lots of channelrhodopsins in one...since current channelrhodopsins tools have low conductance, but also confines channelrhodopsin activation...conductance compared to current channelrhodopsins. Their new channelrhodopsins, called ChRgers, make the brain...

...dedication to resource sharing has led us to develop a searchable and sortable datatable that contains validated...and dCas9-FokI. This table is both sortable and searchable, which can help exclude the data that you are... numeric columns.  The data in any column are searchable and you can search by any part of the data. For...

...in cancer. Yet, the majority of them remain uncharacterized. This represents a considerable barrier to ...Their work culminated in the first functional characterization of over 1,000 mutations, and the group has...are also sensitivity issues when attempting to characterize mutations in a pooled cell population, as mutations... Michael G. Lemieux...

...plasmids is available in an easily searchable format on Addgene’s Michael Davidson Fluorescent Protein Collection...great work of Michael Davidson and his colleagues." Acknowledgments Thank you to Michael Davidson, Michelle...proteins and their use for imaging cell biology, Michael Davidson’s lab at Florida State University has ... the cell, use these markers as your guide. As Michael Davidson told the Tallahassee Democrat in a story...