...everyone who works in a lab struggles with reproducibility at some point. Usually it comes up when a researcher...decides on a new project and begins by trying to reproduce someone else’s result. Then, they hit trouble..... It won’t. When we hear about issues with reproducibility, time and time again, the problem comes down...students and postdocs sitting frustratedly in reproducibility jail and more effective collaboration to drive...Press journals, including Cell Metabolism, Cell Reports and Joule. In September 2017 she moved away from...blog Find more ways to make your research more reproducible Read all of our blog posts on scientific publishing...

...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...to provide ready-to-use viral preps. Here are some of the new viral preps from recent months: Second generation... cells The CRISPR Corner New ready-to-use viral preps   Improved cell-free transcription/translation ...’s lab previously developed such a system in Streptomyces, a prokaryotic genus capable of producing a ...extract.    Graphical Abstract: Schematic representation of cell-free system, capable of producing fluorescent...simple, controlled “one-pot” reaction capable of reproducing useful natural materials.      Moore et al.,...plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE, to identify...

...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...to provide ready-to-use viral preps. Here are some of the new viral preps from recent months: Our selection...strigolactones The CRISPR Corner New ready-to-use viral preps New toolkit for CRISPR-based genome editing in...straightforward workflow for doing gene targeting, reporter generation, controllable gene knockouts, and inducible...CRISPR plasmids are always being added to the repository. To find all of the CRISPR plasmids available...selection of GCaMP8 AAV preps has expanded. Check out the new selection of ready-to-use AAV9 and AAVrg vectors...

...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...to provide ready-to-use viral preps. Here are some of the new viral preps from recent months: Axon targeting...INTRSECT viral preps available! We are expanding our inventory of ready to use viral preps from the Deisseroth...pathways The CRISPR corner New ready-to-use viral preps Mapping antibody escape in SARS-CoV-2 by mutational...interest (Figure 3).   Figure 3: Schematic representation of the MPyV system. Image from Cheah et al....lineage tracing tool captures rare clones that represent 0.001% of the population.  Because indel generation...GCaMP8 vectors in serotypes AAV1 and AAV5. AAV9 preps and more AAV1 versions will be released soon. Visualize...

...viral preps. Here are some of the new viral preps from recent months: New INTRSECT viral preps available...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...generate AID* tagged CRISPR knock-in repair constructs. These repair constructs can be used to integrate...available as a ready-to-use viral prep in AAV1!  New ready-to-use AAV prep from Hongkui Zeng's lab are available...recently contributed a set of plasmids to the repository that will help epigenetic researchers custom ...lab that enables simultaneous gene activation, repression, and editing with a single Cas9-VPR in Saccharomyces...cerevisiae. To control Cas9 activity and influence DNA repair pathways, Amit Choudhary's lab developed a chemogenetic...

...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...CRISPR–Cas9-based Dual-fluorescent DSB Repair) is a double-strand break repair assay for mammalian cells. It's...to provide ready-to-use viral preps. Here are some of the new viral preps from recent months: Expanded...INTRSECT viral preps from the Deisseroth Lab now available. Find all the Deisseroth viral preps here Coming...in a protein.    Cells expressing an RFP reporter plasmid for recombinase activity were exposed ...introduction & resolution of two DSBs in a fluorescent reporter. Find the plasmids here Yongsub Kim’s lab engineered.... Serotypes include AAV1, and AAV9. Find viral preps here pAAV-hSyn-Cre-P2A-dTomato is now available...

...or a pre-made viral prep library. Addgene’s pooled libraries are typically DNA preps, but we do distribute...libraries as pre-made viral preps. If you can’t (or don’t want to) make viral preps in your lab, there are ...are custom viral prep services that can prepare the libraries for you.  Starting from plasmids requires ...pooled library lentiviral prep. B) Usage of a pooled library lentiviral prep for experiments. Pooled library... use! In this post, I’ll walk you through how to prep and transduce your pooled library.  Pooled libraries...verified through NGS sequencing to confirm library representation. Transfection of the DNA pooled library and... library lentiviral preps are generally aliquoted to reduce the number of freeze-thaw cycles needed when...

...making that section of the blot unusable for reprobing. Preparing your western blot Run your gel and membrane... the mild one. Reprobe Once your membrane is clean of antibody, you’re ready to repeat your western blot...able to strip and reprobe your western blot several times — some researchers report up to ten cycles! ...you have nothing to lose by trying to strip and reprobe. In this case, we recommend using mild stripping...proteins through several rounds of stripping and reprobing, maximize your signal by starting with the least-abundant...different rounds of detection. Finally, stripping and reprobing works best with western blots that use chemiluminescent... membrane with antibodies on it if you plan to reprobe! Just as your sample protein is retained better...

...performed using the transfer plasmid, a plasmid encoding Rep and serotype-specific Cap genes, and a plasmid encoding...order to generate high quality preparations. After production, all virus preps are titered and subjected to... containing) AAV particles within representative vector preparations was quantified with electron microscopy...typically prepared using endotoxin-free plasmid purification kits. Resources Browse lentiviral preps available...encoding adenoviral helper sequences. AAV preparations are purified by iodixanol gradient ultracentrifugation...purity and titers adequate for in vivo studies. Preparations are then aliquoted and stored at -80 °C. Titer...on viral genomes isolated from the final AAV preparation. Sequencing results are analyzed to confirm the...

...scientific articles, only 46% could be reproduced. In a recent Nature report from researchers at Amgen, it was... literature was reproducible by their groups. Finally results from the Reproducibility Project: Cancer...have shown similarly disappointing rates of reproducibility. These alarming results are due, at least in...the table in terms of data traceability and reproducibility of research. Electronic lab notebooks (ELNs...ELNs) are here to make lab work much more reproducible, convenient and effective, in both academic circles...through all saved data and automated writing of reports. To be able to understand the situation in depth...keep paper records as well as digital, or for reporting on a project – printing your data out can be required...

...fluorescent executioner caspase reporters. Several apoptosis reporters have been developed, such as the...transfer (FRET)-based executioner caspase reporters. In these reporters, the FRET pair is linked by a caspase...FlipCherry: Red fluorescence protease reporter To add to the fluorogenic reporter toolkit, the Shu Lab also designed...novel fluorogenic reporter that has a large dynamic range and high brightness. This reporter achieves approximately...fluorescence emission. FRET and fluorescence based reporters however have several limitations. First, FRET ...study apoptosis in vivo scientists need a protease reporter that has a large dynamic range and high brightness...Shu Lab from UCSF designed an improved apoptosis reporter, ZipGFP, which utilized the self-assembling split...

...sharing, computational reproducibility, and tools to enable scientific reproducibility. This workshop was ...practice and teach reproducibility An early milestone for the group was the “reproducibility for everyone” ...Scientific Reproducibility Read a blog post on Unique and persistent IDs for improved reproducibility Other...Other resources: Reproducibility for Everyone website How-to-guide for Reproducibility workshops Levelling...protocols.io, organized a panel to discuss reproducibility issues in research. But he realized that it... enough to discuss the age-old problem of irreproducibility in science or even to discuss potential solutions...be an actionable way to tackle widespread irreproducibility. Luckily, there were other academics and like-minded...

...real time, but each of these reporters has its drawbacks. Acoustic reporter genes (ARGs) overcome these...existing bacterial reporters Limited Depth of Signal/Imaging: Fluorescent reporters are not as well suited...fluorescent or bioluminescent reporters usually only detects the signal of the reporter and fail to pinpoint what...signal is coming from.   Acoustic reporter genes (ARGs) Acoustic Reporter Genes (ARGs) are engineered gene...imaging tools The luciferase reporter gene The green fluorescent protein reporter gene Resources on Addgene.org... researchers have developed in vivo bacterial reporters that use fluorophores and luciferases to track...these limitations by using gas vesicle reporters that are detectable by an inexpensive and widely available...

...demonstrated how CRISPR nickases improve homology directed repair rates, and share their design rules for your next...design.  Exploring nickases for homology directed repair The potential benefit of using nickases for HDR...targeting range: using an individual gRNA with WT Cas9, repair levels decrease rapidly 10 bp from the cut site...create a staggered cut - could this system mediate repair throughout the entire region between the nicks?...spots between the nick sites, they observed >20% repair (max 27%) across the 51 nt region using either ... sites tested by a wide margin, exhibiting >20% repair efficiency. Clearly, a nickase strategy can expand...the Addgene Blog Learn about Homology Directed Repair 3 Tips to Improve HDR Efficiency How to Design ...

...Not all plasmid preps are the same. Before purifying a plasmid from a bacterial culture, it is important... on these premises we can classify a plasmid preparation in 3 different ways: Transformation grade DNA...and flawless cloning it is important that the preparation is pure from other nucleic acids (genomic DNA...DNA for each reaction. As a consequence, the preparations for this technique must be larger scale, to ...decide what will be the optimal type of plasmid preparation in terms of quantity and purity.  Additional...

...sending the phages to the clinicians. Creating a repository of phage data and information Since the inception... their role in the phage community by being a repository of phage data and information. “We want to be...particular phage researcher may only know how to prepare phage for one application but not another or they...Directory have the resources or know what it takes to prepare phages for clinical use. There are also different...

...What are Acoustic Reporter Genes (ARGs)? Fluorescent and bioluminescent reporter genes are widely used... to study gene expression in cells. But these reporters have limited use in vivo because they are based...expression in vivo.  But what ultrasound lacked was reporter genes that could produce ultrasound contrast. ...our lab - the Shapiro Lab - developed Acoustic Reporter Genes (ARGs), which are sets of genes that, when...promoter pBAD and a low-copy number origin of replication (p15A) worked best under a wide range of inducer...5202–5209 (2017). Bourdeau, R. W. et al. Acoustic reporter genes for noninvasive imaging of microorganisms... Hurt, R. C. et al. Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors...

...lab team takes over and prepares the sample for entry into Addgene’s repository. For long-term storage ...The lab prepares bacterial glycerol stocks on Fridays. From each overnight culture, we prepare three sets...sent from depositors, bacterial glycerol stocks, prepped DNA ready for our quality control processes - all...missing so that we can make a new tube from a replicate sample. The transformation After the DNA has ...plasmid can grow. As the bacteria divide, they replicate and retain the plasmid. This allows us to make...is contaminated. We use filtered pipet tips and prepare fresh media with antibiotics at each stage. The...Addgene Get help with plasmid transformation, preparation, and analysis from our protocols and molecular...

...development for foreign national researchers and entrepreneurs who are looking to continue their careers in... As an international researcher and aspiring entrepreneur, you have many career options available to you...working under in the lab or has won, awards, media reports, and membership in groups related to the foreign...: You the individual must be named in the media report (NPR, New York Times, respected online sources)...self-driving cars, tissue engineering for organ replacement).  In addition to presenting your expert skills...your work. Immediate steps that you can take to prepare for the green card process Update your CV and ...development firm focused on connecting innovative, entrepreneurial companies with like-minded professionals.  ...

... Figure 4: Reporters in Gene On include a mScarlet reporter, luciferase reporter, and neomycin resistance... editing. eGFP reporters for single base editing by APOBEC-Cas9 Though the ACE reporter quantifies APOBEC-Cas9...DSB in their previous reporter, the Harris Lab created a panel of eGFP reporters to quantify the on-target...editing reporters. Image from St. Martin et al., 2019. The Harris lab used these eGFP reporters to analyze...Transient reporter for editing enrichment (TREE) The Wang and Brafman lab developed a transient reporter for...cells segregated solely by a reporter of transfection, which only report the efficiency of plasmid delivery... functional reporter system to identify and enrich for base-editing activity The reporters mentioned above...