...cloning process, there are also many ways to troubleshoot the cloning experiment. Here are some tips that...cloning strategy you followed. While most of the troubleshooting regarding this step has to be strategy specific...software. Now that you've read our plasmid troubleshooting guide, you hopefully have some new strategies...

...the plasmids we distribute through our quality control processes. Every plasmid we receive is rigorously...repetitive regions (such as LTRs or ITRs), hairpins, or strong terminators. Detailed instructions on other factors...Addgene Blog Get 6 Tips for Analyzing and Troubleshooting DNA Sequence Learn Additional Ways to Verify...  quality control...

...optogenetics: 1) electrophysiology and 2) behavioral experiments. I’m not an electrophysiologist so I cannot...to hook up your lasers and not have the proper controls or a proper behavioral readout that allows you...behavior, you don’t necessarily need to use electrophysiology to prove that your optogenetic manipulations... (though a reviewer will probably ask for electrophysiology data too but that’s what collaborators are...not be an appropriate test.  Of course, proper controls are required and include, for example, confirmation...Carolina) that offer his constructs. Importantly, control viruses (not expressing the opsin) are also available...into the correct direction and expressed (under control of a generic promoter). A DIO opsin allows cell...

..., VVDfast to control recruitment of an “anchor” to microtubules and iLID-SspB to control the recruitment...reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular...Optogenetics gives you the power to control cells or organisms with the flip of a switch. You may be ...with popular light-sensitive ion channels used to control activities like neuronal signaling — think of a... a few recently-deposited optogenetic tools to control the cytoskeleton and cell membrane dynamics. First...component of the cytoskeleton – actin? Actin dynamics control cell morphology, movement, and more, often driven...specific Rho GTPase/GEF pair (Ridley, 2016). Taking control of a RhoGEF is a great way to stimulate and study...

...rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but often at ...synapses.)  In this post we’ll describe the AAV retrograde serotype (AAVrg) and how it enables scientists...visualize neurons that send information to a region (retrograde tracer, Fig. 1A), and another to visualize neurons...upstream from the axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels...tracers, but finding a virus that works well for retrograde tracing can be difficult. The in vivo workhorse...adeno associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes. Some ...directs high-level transgene expression, and has retrograde functionality to monitor and map neurons (Tervo...

...developing a new fluorescent protein tool that responds strongly to magnetic fields (Frank Hayward et al., 2024... of a magnetoresponsive fluorescent protein. Controlling an optogenetic or fluorescent tool with magnets... 2016), and some previous attempts at magnetic control of biological systems (Wheeler et al., 2016; Duret...al., 2019). But the field has sometimes seen controversy, and the interactions of organic molecules like...science in the open The York Lab operates with a strong commitment to exploration and collaboration. Andrew...says, noting that his team regularly shares troubleshooting tips and works with other labs to help them...Avants, B. W., & Robinson, J. T. (2019). Magnetic Entropy as a Proposed Gating Mechanism for Magnetogenetic...

...recorded events) based on their expected size. The strong signals close to the graph’s origin mainly stem...

... Viruses of the Retroviridae or Retrovirus family, which includes the gamma-retrovirus and lentivirus ...using retroviral vectors in your experiments. Gamma-retroviral vectors Popular gamma-Retroviral Transfer...genome-wide experiments. Gamma-retroviral vs. lentiviral vector systems Gamma-retroviral vectors have a relatively...cells. Find tips on using gamma-retroviral vectors in our Gamma-retrovirus guide and find popular packaging... codon-optimized Cas9 under the control of Murine Stem Cell retrovirus promoter for mammalian gene knockdown...plasmids for transcriptional inhibition. Retroviral Plasmid Kit Retroviral Barcoding Library (Kit # 1000000097...multiple genes via infection with a single ecotropic or amphotropic MuLE lentivirus provides significant genetic...

...insertions require complicated, often costly in vitro assembly methods. A new method, REPLACR-mutagenesis...mutagenesis methods Current SDM methods rely on in vitro assembly. To make a point mutation, you design two...Trehan et al. wondered if, instead of relying on in vitro assembly, they could make the E. coli do the work...must PCR amplify the whole plasmid, which could introduce mutations. Using commercially available high-fidelity...-mutagenesis also requires making your own electrocompetent cells in which the phage recombination machinery...since you skip the steps of gel purification and in vitro recombination. Across all modifications described...Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes Yes DpnI Digestion Yes Yes ...

...mutations in dystrophin, a protein essential for proper muscle function. Without functioning dystrophin, an individual... condition known as Becker muscular dystrophy (BMD). Dystrophin’s size makes it difficult to deliver via...through NHEJ, will restore the dystrophin reading frame. gRNAs bind in intronic regions, so low-frequency ...editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science. 2015 Dec 31... CA. Correction of dystrophin expression in cells from Duchenne muscular dystrophy patients through genomic...editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy. Nat Commun. 2015b Feb...Our Special Collections Pages Duchenne muscular dystrophy: The most common inherited disease The genetic...

...always include proper controls like uncut DNA and the proper ladder. These controls help you properly visualize...sequencing the chromatogram is your visual control. And, like all controls, missing out is a big mistake. Chromatograms...Chromatograms come to the rescue for analyzing and troubleshooting your DNA sequencing results Above (top right...repeated. Guidelines to help with DNA sequencing troubleshooting and analysis 1. You can use any of the following...70-75 of the read (see image below), so I would strongly recommend using a silica spin column. They can...most out of your DNA sequencing results and to troubleshoot any problems that come up. Good luck analyzing...

... in extracurricular activities (EA). EAs can: Introduce new perspectives in science and engineering Looking...that your project is an innovative solution with a strong impact, or a journalist does not understand the...

...spatial control. Optogenetics uses genetically encoded tools, such as microbial opsins, to control cellular...genomic loci Photochemical Control Hemphill et al., 2015   Nitrobenzyl-caged lysine Cas9 K866PCK mutant...tools allow scientists to use light to externally control the location, timing, and reversibility of the ...process. Read on to learn about the various light-controlled CRISPR tools available to researchers - some ...published in early 2015 focused on using light to control transcription. Two separate labs, Moritoshi Sato...gene expression while imparting spatiotemporal control, reversibility, and repeatability. The system developed...expression of the human ASCL1 gene could be spatially controlled in a reversible and repeatable manner. With their...

...Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use electroporation to ensure...library, Addgene usually does four to eight electroporation reactions using a total of ~400ng of library...

...loading controls are equally strong, so the normalized value simply reports which POI band is stronger. The...loading control is not perturbed by the experiment The loading control is supposed to control for, well...western blots benefit from a positive control — a loading control — that you know is present in every lane...transfer. Figure 1: A loading control is a type of positive control necessary to demonstrate a POI’s...loading control: the intensity of each POI band is divided by the intensity of the loading control in the...with loading controls. Under each lane, the fraction of the POI band over the loading control is shown. ...loading control. First, let’s explore why these assumptions are key to using loading controls accurately...

...choice for targeting astroglia. It has additionally been reported to have tropism for lung, liver, vascular...as in-stock preparations. AAV retrograde (AAVrg, also known as AAV2-retro) We’ve covered AAVrg in a previous...following table focuses on tissue tropism in mice in vivo. Though tropism depends on many variables, we suggest...the opposite: AAV serotypes with varying tissue tropism, allowing them to target particular cell types....commonly used serotypes in this post. However, tissue tropism varies both between species and even between strains...choice for AAV pseudotyping. It has broad tissue tropism, including liver, muscle, lung, CNS, kidney, and...service.  AAV9 AAV9 has relatively broad tissue tropism and high transduction efficiency, and is an excellent...

...The widespread use of γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted...flexibility to pseudotype γ-retrovirus using different envelopes to modify tropism. Briefly, different envelope...either an ecotropic envelope, Phoenix-ECO, (for infection of mouse and rat cells), or an amphotropic envelope...prototypical gamma-retrovirus.    Frequently asked questions about γ-retroviral vectors How do engineered...MoMLV and MSCV for γ-retrovirus; HIV for lentivirus). Additionally, γ-retroviruses can only transduce dividing... Are γ-retroviruses self-inactivating (SIN)? They can be. Plasmids that encode SIN γ-retrovirus lack most...blog Viral Vector Elements The Retroviral Life Cycle Popular Retroviral Vectors and Their Use in Research...

...and developed the SciNote Electronic Lab Notebook. Implementing an electronic lab notebook starts with ...should have functionalities such as: electronic signatures, electronic witnessing and audit trails. How ...write things down when doing scientific research? Electronic lab notebooks and researcher's needs The familiar...traceability and reproducibility of research. Electronic lab notebooks (ELNs) are here to make lab work...This includes financial outlay, staff hours, troubleshooting, and the fact that long-term use is likely ...traceable. However, switching from paper to an electronic lab notebook requires time and knowledge. In ...recent article, we translated our knowledge of electronic lab notebooks into 9 questions that will help...

...highest quality, we undertake a rigorous quality control process. In 2016, we launched our viral service...our scientists perform all the standard quality control assays for AAVs. These steps include measuring ...Addgene is also conducting more rigorous quality control, including next generation sequencing (NGS) of ...VGS). Why should you care about viral quality control? AAV vectors are produced by scientists - not robots...Lecomte et al., 2015). This higher level of quality control is recommended but not yet required for clinical-grade...addgene.org to answer your questions about our quality control process! The images in this blog post were created...2011) Vector Characterization Methods for Quality Control Testing of Recombinant Adeno-Associated Viruses...

...Nicola Patron is Head of Synthetic Biology at the Sainsbury Laboratory, where she often feels more like...that go on between plants and their pathogens. As Patron explains it, her work has always been focused on...Modular Cloning (MoClo) Kit. What does it include? Patron: Sainsbury has a mandate to share and educate and...Addgene: Will the MoClo tools work in any plant? Patron: The MoClo kit is entirely for dicots, not grasses...tips for those looking to get started with MoClo? Patron: The parts will be familiar to most plant scientists...there other tools you are working on for plants? Patron: Our lab and others at the Sainsbury are working... of engaging about science in that way for you? Patron: I started my Twitter account recently inspired...