...For example, the Levin lab from Tufts University has used Arch (green-light activated proton pump) and...Allen Discovery Center. The future of this field has a high potential for the advancement of research ...based on the fusion of the voltage sensing domain of a Ciona intestinalis voltage sensor and a mutated version... reporting and genetically encoded fluorescent voltage indicators (GEVI) are ideal tools to track bioelectric... 2018) available at Addgene: MerMAID2 - A metagenomically discovered marine anion-conducting channelrhodopsin...channelrhodopsin used as a sensitive fluorescent voltage indicator with near-complete desensitization in continuous...Endogenous bioelectric signaling networks: exploiting voltage gradients for control of growth and form." Annual...

...top of the resolving gel. A stacking gel typically has a different ionic strength and lower pH and acrylamide...Choosing your primary antibody Once the membrane has been blocked, it is stained with a primary antibody...the vendor’s website and choose an antibody that has been validated for immunoblotting. Different immunoassays..., cytoplasm and nucleus.  We hope that this blog has provided you with a good overview of the western ... of the gel. For small proteins, use a higher percentage of acrylamide to increase migration resistance...improve separation. For large proteins, reduce the percentage of acrylamide. To further improve resolution,...

...community has access to all of the latest resources? In response to this challenge, Addgene has launched... why recent work in the field of cancer research has focused on picking apart the complex cellular pathways...at Vanderbilt University in Nashville, Tennessee has already demonstrated that a number of novel small...ensues, traditional chemotherapies have taken advantage of the universally-dividing behavior of cancer...

...Not the case, as I’ve discovered.) As someone who has now seen both sides of the postdoc experience, I’... lab is closely involved with clinical trials or has ties to industry, the drug discovery process can ...Genentech, will not. The point is that each company has a unique approach, and some might suit your needs...currently a Postdoctoral Scientist at AstraZeneca. She has a background in cell and developmental biology and...number of steps, decisions and people involved is staggering. The process is complicated. If you’re interested...

... ddPCR.) qPCR Quantitative/real-time PCR (qPCR) has been used for copy number assays for years. qPCR ...and quencher to quantify specific amplicons.  Sybr has high background noise, due to the large amount of...AT-rich DNA and the length of the amplicon and it has a higher rate of false positives (Colborn, 2008; ... used to accurately call copy numbers, though it has been shown to identify trends correctly (Muhamad,...Muhamad, 2011;  Providenti, 2006). Its biggest advantage over Taqman is price point, which should be carefully...

...attributes (which turned into www.fpvis.org).  That page has been very useful over the years, and when researchers...system that allows users to see when a given page has unmoderated changes and to view previous versions... online collections of FP spectra. Every protein has a page with all the information in the database relating...of FPbase FPbase is still very much in the early stages of development, and there is plenty of room for...

...birth several weeks previously. While this claim has yet to be independently verified, the watching world...Kingdom’s Nuffield Council. While the Nuffield Council has recently published a report on the social and ethical...considerations, in making sure that the research has been adequately proven to be reasonably safe and ..., in vitro research, animal models, and several stages of human clinical trials. This model also demonstrates...

...easy. Most of our equipment has been donated by local companies and BosLab has grown into an organization... biologist with a Ph.D. from UNC-Chapel Hill and has extensive experience targeting RNA with small molecules... entrepreneurs using our space to perform early-stage experiments. For example, Dave Grawoig is using ...

...booklet. What has been the most surprising thing about becoming a PI? Tom Ellis has a one word answer...prepared me for what a PI without a personal assistant has to deal with. My inbox currently stands at about ... build a strong, collaborative team. And take advantage of the many resources available from your institution...

...Find Empty Backbones at Addgene Each approach has advantages and drawbacks, which are summarized in the ...for the presence of an insert. Traditionally this has been done with restriction enzyme digest; however...insert-specific primers but also tell you whether the insert has the correct directionality. Verifying the insert ...and are more likely to work in a PCR reaction that has bacterial debris. Use a positive control. A good...

... population level, but each individual cell only has a maximum of one gene knocked out. In this genetically...containing up to around 200,000 different gRNAs. It has been suggested that researchers should use ~400X ...than gRNAs in a screen, to ensure that each gRNA has a good chance to contribute to the screen. In the... If you are lucky and find that a druggable gene has a central role in your system, you could majorly ...guest blogger Fredrik Wermeling! Fredrik Wermeling has a research group at the Centrum for Molecular Medicine...Medicine, Solna, Karolinska Institutet, in Sweden. He has a particular interest in using molecular tools to... differentially expressed genes can be the next stage in making sense of such data. These targeted screens...

...restriction endonucleases. A given prokaryote typically has genes encoding one or a few restriction modification.... Using the same example plasmid as above (which has the XbaI site blocked by methylation), the figure...newly synthesized product from a site-directed mutagenesis experiment? The answer to both questions is the...to methylation if the enzyme's recognition site (TCTAGA) is preceded by GA or followed by TC. As shown ...cleave DNA. DpnI is often used for site directed mutagenesis. During this process, incorporation of a desired...Troubleshoot Your Sequencing Results Use REPLACR Mutagenesis to Mutate Your Plasmids Resources on Addgene.org...

...the opsin you use); the efficacy of optogenetics has already been proven. Therefore, a concrete, well ...scientists who pioneered the development of optogenetics, has genetically manipulated a number of the opsin genes...kinetics of opsin activity/inactivity. Dr. Deisseroth has also generously made many of his optogenetic constructs...stereotaxic surgery (see videos below). The Deisseroth lab has developed many of these AAV constructs with various...promoter of the cre recombinase while still taking advantage of the robust expression via the promoter in the...stereotaxic frame with a digital readout. The advantage is you can “zero” your coordinates at bregma which...engineered to express various opsin genes [7-9]. The advantage of using these lines is, if one is crossed with...

...extension PCR. Although each type of cloning has its advantages, most scientists still encounter several ...science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis Perrakis. "Recombinant cloning...Plasmid Cloning by PCR Perform Site Directed Mutagenesis by PCR Catch up on Your Plasmid Background with...

...partnership was fruitful and a first version of the study has just been uploaded to the preprint repository BioRxiv... platform; while they are a minority, their help has been essential to push certain projects. The idea...year on, our biggest lesson running the platform has been the realization that there is a great untapped...Crowdfight is also open to other topics.  In this new stage of the project we are focusing on what we call “...

...method While the hybrid dual vector system has a slight advantage over the overlapping and trans-splicing...Adeno-associated virus (AAV) has many features which make it a great viral vector, but its packaging ... Overlapping  Overview: The overlapping strategy has a region of homology, typically ~400-1400 bp long...Drawbacks:  Generally an overlapping split vector has equal or higher transgene expression when compared...efficiency of transgene expression from split vectors has been inconsistent. A greater understanding of basic...

... editors be close at hand? Once again, this post has a lot of acronyms, so we've included a glossary at...or C substrates. The HuidaGene Therapeutics team has deposited their editors, called gTBE and gCBE, with... abasic site. The same protein (or its homologs) has more than one name. Indel Insertion/deletion A ...this promising start, the team used rational mutagenesis to produce the adenine transversion editor AYBEv3...and ABEs, they could be useful for saturation mutagenesis, and they provide a strong proof of concept for...empty space. Over several rounds of rational mutagenesis, the team progressed to gGBEv6.3, which was capable...

...in vivo scientists need a protease reporter that has a large dynamic range and high brightness. The Shu...in the midgut of Drosophila, an observation that has been difficult until now (Zhang et al., 2019). Conclusion...Conclusion FlipGFP is a novel fluorogenic reporter that has a large dynamic range and high brightness. This reporter...minutes (Cabantous et al., 2013). The Shu Lab took advantage of this and came up with a system where fluorescence...

...-homologue recombination was released. This blog has not been updated in response to this paper. Any hint... hint of CRISPR editing in human embryos has been met with a storm of media coverage. But the paper published... the edited embryos proceeded to the blastocyst stage at similar rates to untreated controls. Ma et al...results in fewer eggs. However, the potentially staggering costs of such a treatment on top of the expense...limited their whole genome sequencing to a small percentage of edited embryos. Since the embryos produced...had few predicted off-target effects, but this advantage likely won’t transfer to all genes we’d like to...worthwhile for a given disease if it increased the percentage of disease allele-free embryos to only 60%? Unfortunately...

...prime editing By Emily P. Bentley The David Liu Lab has made two recent deposits focused on optimizing prime...collaboration with the Vallabh/Minikel Lab, the Weissman Lab has developed a new epigenetic editor, Coupled Histone...subtypes, and a familiar challenge for anyone who has done histology on brain tissue is identifying the...magnetic beads through an assembly of biotinylated SpyTag-SpyCatcher linkers and spacer proteins ending with... single alpha helix) and target capture module (SpyTag-GFP nanobody) assembled on beads. C) Micrograph...methods such as chemical crosslinking or tilted-stage imaging. A truncated form of a tardigrade LEA, RvLEAMshort...Release of Methyltransferase (CHARM), with several advantages over the earlier system.  Rather than overexpressing...