...Minisymposium on Reproducibility. The minisymposium focused on solutions to reproducibility issues in the...own talk on reproducibility at Addgene and here I summarize what I learned both in preparation for my talk...ways of defining scientific reproducibility. For a study to be “reproducible” does not necessarily mean...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...concepts. However, rates of reproducibility for preclinical studies, as reported by drug companies attempting... the Reproducibility Project: Cancer Biology have produced similarly low rates of reproducibility. Failed... enterprise towards greater reproducibility. Cause 1: Inability to repeat previously performed experiments...

...site-directed mutagenesis by recombineering.” Sci Rep 6:19121. PubMed PMID: 26750263. PubMed Central PMCID: PMC4707547...cost. Read on to find out how to replace your SDM method with REPLACR (Recombineering of Ends of Linearized...al., REPLACR-mutagenesis’ median efficiency is 75%. If these numbers hold up across labs, REPLACR-mutagenesis... time. Let us know how REPLACR-mutagenesis works in your hands!     REPLACR-Mutagenesis Megaprimer ...costly in vitro assembly methods. A new method, REPLACR-mutagenesis, harnesses the power of bacterial recombineering...are then transformed into E. coli to undergo nick repair. This method can work well, but poor template digestion...secondary structure or too much sequence similarity. REPLACR-mutagenesis: Letting bacteria do the work If you...

... Created with BioRender.com   Replication (rep) gene The rep gene encodes four proteins that are required...domains, all four Rep proteins share a common helicase and ATPase domains. To function, the Rep proteins form...required for viral genome replication and packaging: Rep78, Rep68, Rep52, and Rep40. Rep gene expression is ...respectively, produce the large (Rep78 and Rep68) and small (Rep52 and Rep40) Rep proteins. Alternative splicing...oligomers of Rep 78/68 or Rep52/40. Rep78/68 play a key role in AAV genome replication. Replication initiates...packaging plasmid aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (cap...C-terminal regions of the Rep proteins. While the unique N-terminal domain of Rep78 and Rep68 contains a DNA-binding...

...References ASAPbio, Preprint Resource Center: https://asapbio.org/preprint-info ASAPbio, Preprints and Rapid Communication... the possibly repeated rounds of rejection and resubmission to other journals. Preprints allow authors...Transparency Another major benefit of preprints is their openness. Preprints can be read freely by anyone and...Lancaster et al. (2018) Of course, preprints don’t replace journals and other modes of scientific ...of the benefits of preprints. Some worry about faulty work being posted as preprints, assuming that peer...the manuscript prior to submission. Preprints at Addgene Preprints are all about sharing scientific knowledge...described in a preprint. In fact, over 1,500 plasmids in our catalog either list a bioRxiv preprint as the source...

...reduce binding with Rep proteins and mutations in the trs site reduce Rep nicking. Since Rep proteins are important...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs...4-nucleotide Rep binding element (RBE) that serves as a binding site for Rep78 and Rep68 to initiate replication... replication and a terminal resolution site that serves as the target site for Rep proteins (Savvy et ...inverted terminal repeats reduces rAAV productivity but increases transgene expression. Sci Rep, 7(1), 5432....-C’ regions) and forms a T-shaped structure. The Rep binding element (RBE) and terminal resolution site... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...

...detectable by two different tags: a reporter and an HA tag. The reporter comes in three varieties: HRP, Spaghetti...independent of the reporter. The only caveat is that HA staining is weaker than reporter staining. Picking...Picking the right reporter for your RANBody While all three reporters stain cultured cells and tissue sections...Monster, and Chicken IgY Domain RANbody Reporters. Reporter Detection Method Uses Pros Cons Antigens...genetically tagged and instead are conjugated to reporters (i.e. fluorescent dyes, HRP, etc. ) after purification... is a RANbody? A RANbody is a fusion between a reporter and a nanobody that’s encoded on a plasmid. A ...sections, some reporters are better suited for particular applications. Read on to learn more about each...

...means that reproducibility is limited in two ways – only data inside the ELN is reproducible, and only ...source data repositories Over the past decade or so a number of general purpose repositories have been ...value the repository integrations, because when RSpace documents are deposited into the repositories, links... and/or attempt to reproduce research relating to data deposited into a repository access to that part..., but some ELNs provide better support for reproducibility than others. Unfortunately, most ELNs are designed...access the ELN have the ability to attempt to reproduce the research that produced the data. In this post...capabilities an ELN needs to facilitate true reproducibility. The following are critical: (1) connectivity...

...ve found helpful for my interviews in industry. Prepare stories to tell in your interview The STAR interview...interview has started. Instead, I preferred to prepare stories to answer interview questions. Story telling...starting place for drafting my stories. In total, I prepare 5-6 stories for an interview, since one story usually...stories helped me remember them for the interview.  Prepare a list of questions to ask your interviewers You... as much as they are interviewing you, so come prepared with questions to ask. With a quick Google search...and company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking...deliverable. Find 10 steps to a perfect science talk Be prepared for potential technical difficulties. Have your...

...interconvertible Drosophila overexpression transgenes. Sci Rep 12, 3835 (2022). https://doi.org/10.1038/s41598-022...transcription is ON. Once a co-repressor protein binds the repressor protein, the repressor protein can bind to ...the GAL80 repressor, adds a repressible element to this binary system (making this a repressible binary ...post! How are repressible promoters regulated? Like inducible promoters, repressible promoters can be...Positive Repressible: An activator protein is bound, turning transcription ON. When a repressor binds the... transcription is turned OFF. Negative Repressible: A repressor protein is present, but cannot bind the...both natural and engineered repressible promoters. Chemically repressible promoters Tet-Off In its native...

...the origin of replication/replicon. The replicon is comprised of the origin of replication (ori) and all...ori is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must to survive... cells. The replicons of plasmids are generally different from the those used to replicate the host's ...   Types of origins of replication There are lots of origins of replication out there so, for simplicity...gene for replication (reference). **F1 is a phage-derived ori that allows for the replication and packaging...readily at lower temperatures and allows the replication machinery room to come in and get busy making...derivatives), pSC101, R6K, and 15A. Not all origins of replication are created equal. Some will produce many plasmid...

...notes) Any Data repository Only accepts data for antibodies in Addgene’s repository  IBEX Knowledge-Base...Data repository See blog post mAb3D Atlas Mouse and human brain  IHC Data repository See...there are websites that can help: Antibody data repositories, which share validation data, and antibody search...using antibody search engines and antibody data repositories to find an antibody for your experiment. Search...available antibodies from many vendors, while data repositories share validation and experimental data to help...familiar with in Table 1.  Table 1: Antibody data repositories and search engines Website Targets Application... Notes Antibodypedia Any Any Data repository   YCharOS Human proteins Immunoblot (...

...own replication. Like RepA, RepZ helps recruit host initiation proteins to the plasmid ori. In this system...plasmids rely on their host cell's replication machinery in order to replicate—but not always to the same extent...described in our previous Origin of Replication post, DNA replication is initiated at the ori and may or...consider the replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...review how E. coli normally replicate their chromosomal DNA. Chromosomal replication in bacteria is carefully...entire round of replication. Initiation is the primary control point of DNA replication in bacteria, so... the replication forks reach each other on the opposite end of the chromosome, ending replication.   ...

... CRISPR fluorescent reporter assays to quantify targeted Cas9 cleavage, and repair through homology directed...directed repair (HDR) and non-homologous end joining (NHEJ). In these reporters, editing typically results... CRISPR reporters in the near future.  SRIRACCHA (stable, but reversible, integrated reporter for assaying...histone)-GFP reporter into the genome of mammalian cells.  Figure 1: The SRIRACCHA reporter construct...while cells containing the reporter gene that was cleaved by Cas9 and repaired via HDR using the supplied...Find GEmCherry2 reporter plasmids at Addgene Overall SRIRACHA and GEmCherry2 reporter systems provide ...integrated surrogate reporter for assaying CRISPR/Cas9-stimulated homology-directed repair. Journal of Biological...

...homology directed repair, one of the repair pathways of DSBs, with a focus on repair for genome modification...homology directed repair fix DNA double-strand breaks?  In eukaryotic cells, repair of DSBs occurs primarily...sequence as a template for repair and is thus the more accurate mechanism for DSB repair. The process is generally...then resolve to complete the DNA repair process Homology directed repair pathways HDR mechanisms can be...double-strand break repair (DSBR), synthesis-dependent strand-annealing (SDSA), and break-induced repair (BIR). The...for the repair of breaks that lack a second end to enable DSBR or SDSA.   Homology directed repair and genome...Non-Homologous End-Joining (NHEJ) and Homology Directed Repair (HDR). NHEJ is the canonical homology-independent...

...Neuromodulators like dopamine and norepinephrine have important functions in the brain but have been ... activity. In 2019, the first generation of norepinephrine sensors was developed, named GRABNE, which ...which helped researchers visualize norepinephrine (Feng et al., 2019). But this first generation of tools ...limitations by developing and depositing two new norepinephrine indicators: nLightG and nLightR (Kagiampaki... using ColabFold (Mirdita et al., 2022). B, Representative images of HEK293T cells and neurons expressing...indicators are more sensitive to and selective for norepinephrine and faster to respond than GRABNE. While GRABNE... provide inhibitory feedback in response to norepinephrine release, and clinically-used drugs targeting...

...freshly collected lentiviral prep may be significantly higher than the same prep that has been stored in the...painstakingly cared for your packaging cell line, prepped your DNA, transfected and harvested your lentivirus...determine the amount of virus you actually have in your prep. Taking time to properly titer your virus will not...qPCR for viral RNA. Measuring p24 levels in a viral prep is straightforward and rapid as there are several...universal primers can be used to titer any lentiviral prep that shares that specific feature. Primers targeting...transfer plasmid was used in the transfection; if preparing several different lentiviruses in parallel, this...overestimate the amount of infectious virus in a prep as the measurement will include defective particles...

...much more about cellular DNA repair pathways. An emphasis on cellular repair post-CRISPR cleavage When I...just how complicated DNA repair pathways are. I fully admit to being a DNA repair newbie - diagrams of resection...patterns with ssDNA repair templates. Their work demonstrates that different repair template strategies...Blugene and I represented Addgene at the recent Keystone meeting on Precision Genome Editing with Programmable... to CRISPR nucleases, but with a session on DNA repair. What stuck most in my mind from that session were... complexity and challenges of homology directed repair (HDR) are echoed in the amount of interest in base... to #KSgenome and seeing the intricacies of DNA repair has emphasized how essential these studies are ...

...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...

...through a repository, they remain accessible during these times. Centralized reagent repositories, also known...dedicated facilities and manpower that a repository provides. Repositories around the world facilitate the rapid...to be deposited into repositories, and financial support to maintain repositories’ capacity for storing... the scientific community can support repositories. Repositories are essential for the advancement of ...original sources. Figure 1: Biological repositories that distribute mouse strains, plasmids, cell...critical for pandemic research. Image from Reproducibility for Everyone. Case study 1: Cell lines Many...Culture Collection (ATCC) is a private, nonprofit repository for a variety of materials for life science research...

...brought issues in reproducible research to the forefront in the so-called “reproducibility crisis.” Results...after credible attempts to replicate major findings could not be replicated by other researchers.There...published research is difficult to replicate.  Why is replicating previous research findings so difficult...can be difficult to replicate. Unclear methods Until someone attempts to replicate the results of another...over the course of the Reproducibility Project: Cancer Biology, where reported methods may not include... registered reports, the gold standard for testing hypotheses Finally, Registered Reports combine all ...attention on the results instead of the methods. Unreported flexibility and questionable research practices...