...family of viruses with broad host range and tissue tropism (Borkenhagen et al., 2019). AdVs are non-enveloped...kilobases; compared to only 4.7 kb for AAV, 9 kb for retroviruses). The tradeoff is that adenoviral vector expression...proteins and downstream cellular events, all recruit strong innate and adaptive immune responses that inactivate...and rapidly caused the death of adenoid cells in vitro (Rowe et al., 1953). While overt death of the targeted...than for other vector types, we hope that this introduction gives a sense of what AdVs are about – and we...References and resources References Bauzon, M., Castro, D., Karr, M., Hawkins, L. K., & Hermiston, T. ...Applications Adenoviral Vector Production and Troubleshooting Viral Vectors 101: Types of Viruses...

...specificity control, secondary antibody specificity control, label control, and cross-reactivity control when...helpful to focus on something controllable, like experiments (and their controls!). This blog post will discuss... ins and outs of controls for biological experiments, starting with general controls and then moving on... of controls and be able to use this information to design thoughtfully and thoroughly controlled experiments...experiments.  Broadly useful controls Positive and negative controls Most biological experiments require...positive and negative controls to ensure proper interpretation of results. A positive control is generally a ...serum or isotype-specific control (Hewitt 2014, Alexander 2018). In this control, the primary antibody is...

... strip cycle. Troubleshooting the troubleshooting There’s many different troubleshooting and optimization...blot optimization and troubleshooting.   Before you can optimize or troubleshoot a western, you’ll need...time troubleshooting an assay that’s fine with antibodies that are not.   Table 1: Troubleshooting a western...and may require multiple runs to optimize or troubleshoot.   Before you start… Mark your membrane! Membranes...document it.   Did it work? Save yourself time and trouble by confirming your transfer worked with Ponceau...box to visualize proteins after transfer. Troubleshooting The two most common (non-technical) issues ...your membrane size is probably the easiest troubleshooting step to take. If you’re not sure if you need...

... challenge with using AdV is that it triggers a strong immune response after in vivo administration, which...loss of transgene expression (Interestingly, the strong immunogenicity of AdVs is what makes them ideal...reference 3. Production, amplification, and quality control of 2nd generation AdV     AdV Production in HEK293...looking at absorbance at 260nm using UV/Vis spectrophotometry. An absorbance of at 260nm corresponds to...reference material should be used as an internal control (ATCC#VR-1516) until the assay is validated. Infectious...Adenoviral delivery of CRISPR/Cas9 5 tips for troubleshooting viral transductions Resources on Addgene.org...

...include controls to show that your antibodies are binding specifically. For positive controls, aim to ... and isotype controls are also commonly used in IF experiments. Precisely which controls you perform will...for some background on fluorescence, try the Introduction to Fluorescence Microscopy video from iBiology...been manipulated to overexpress it. For negative controls, use samples in which the target is known to be...research antibodies, protocols, and appropriate controls and you will be well on your way to performing... K, Mareninov S, Diaz MFP, Yong WH (2018) An Introduction to Performing Immunofluorescence Staining. In...10.1007/978-1-4939-8935-5_26 Stuurman N. (2012) Introduction to Fluorescence Microscopy. iBiology. https:...

...detection. You’ll also want to use the appropriate controls for your experiment to account for potential non-specific...quantitatively if you also load the gel with appropriate controls for normalizing the protein concentration. To ...plasmid encoding GFP. Actin, a common internal control for Western blots, shows that a similar concentration...biosensor biorecognition elements. Biosensors and Bioelectronics 85:32–45 . https://doi.org/10.1016/j.bios.2016.04.091...

...Many researchers find themselves plagued by troubleshooting a virus-based experiment at one point or another...will share five hail Marys that have helped me troubleshoot some technical obstacles when working with virus... enzyme digest and compare it to some reliable controls. 2. Viral stocks may be sensitive to freeze thaw...instead of storing it briefly at -80 °C. If troubleshooting, the safest option may be to avoid freezing... to a target cell by reducing the repulsive electrostatic forces between these two negatively-charged ...not work if the virus does not have the proper tropism to infect the packaging cells. Another method of..., and Martin L. Yarmush. "Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent...

...replicate controls.  Transfection controls: empty vector and internal control A transfection control measures...Negative controls: untreated cells, empty vector control, and non-targeting control Negative control conditions...lacking the proper controls are invariably inconclusive and unreliable. Proper controls provide the constant...about the various controls that can be used for plasmid-based experiments. What are control plasmids? Generally...experiment, delineate its requisite control plasmids, and discuss why these control plasmids are critical for the...use of proper control plasmids would address and answer these questions. Types of control plasmids Part...the target cells. One transfection control is an empty vector control; specifically, the plasmid without...

...pMSCV-pBabeMCS-IRES-RFP IRES Retroviral pMSCV-IRES-YFP II IRES Retroviral pCMMP-MCS-IRES-Puro IRES Retroviral pEF1a-...bidirectional promoters, or the creation of bicistronic or multicistronic vectors. Unlike promoters which will...upstream cistron will have a few additional residues added to the end, while the downstream cistron will ... Plasmid Name Multicistronic Element Expression Type MSCV-IRES-EGFP IRES Retroviral pMSCV-pBabeMCS-IRES-RFP...transcripts for each gene that is expressed, multicistronic vectors simultaneously express two or more ... so in this blog post we’ll cover basics of multicistronic vectors: why they are useful, how they work... and how to get started with them. Why Use multicistronic vectors? Detecting cells that are expressing...

...relevant information for planning, executing, and troubleshooting your experiments. For example, if you’re very...

...familiar with the basics of controls in experimental design, you’ll need a few controls specific to flow as an...nonspecific binding. Flow specific controls The three types of flow cytometry controls we’ll discuss are single ... may need to include additional controls like FMO or isotype controls, which we’ll describe a bit later...color control for every fluorophore you intend to use in your panel. Using single color controls On a ...your single color controls may not be accurate for your samples. The single color controls should be used...plots for a single color control. A) A flow plot showing a single color control for the APC channel. B)...likely to require FMOs. Isotypes Isotype controls are negative controls that allow you to determine what level...

...The Retroviridae (commonly called retrovirus) family — of which HIV is a member — may seem like an unlikely...molecular biology. “Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses — that have...have been developed into retroviral vectors and 2.) a subclass of retroviruses called the lentiviruses (...foundation for producing and using retroviral vectors effectively. Retroviral structure   Figure 2:..., as the viral particle emerges. Retrovirus life cycle Retroviruses go against the grain of the Central...unlikely candidate to use as a viral vector, but retroviruses have been developed into some of the most widely...Lentiviruses are sometimes referred to as “complex retroviruses ” (Cullen, 1992) because of their more complicated...

...work, although it’s less effective in vivo than in vitro. On the other hand, if you’re interested in expressing...and serotype selection. Promoters and serotype control, respectively, cell-specific gene expression and...viral infection. Cre or Flp dependent switches control gene expression via switches placed in your cell...updates from Addgenies Karen Guerin and Susanna Stroik. Didem Goz Ayturk is a Postdoctoral Fellow in Connie...System: Towards Gene Therapy For Duchenne Muscular Dystrophy. InTech. doi: 10.5772/53023 Veldwijk, Marlon R...

...if the two proteins form a complex. To help troubleshoot your experimental setup as a potential source...FRET measurements and serve as a type of positive control. In the case of an unfavorable donor-acceptor orientation...

...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...

... language you’ll see throughout the posts and introduce *the* question underlying all communications efforts...

...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...

...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...

..., 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...

... 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...

...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...