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...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...an intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or...cell. The cell then uses its own machinery to replicate the viral genome and proteins. This is, for the...before being translated into proteins. RNA viruses replicate their genomes using a variety of RNA or DNA intermediaries... bacteria and eukaryotes, which use a binary replication process whereby one cell copies itself and “splits...

...are used across species: replication, selection, and expression. Replication It’s usually not enough to...chromosome has a single origin of replication (ori), where DNA replication begins, so a bacterial plasmid...Eukaryotes are a bit more complicated. Yeast vectors may contain an autonomously replicating sequence (ARS), a...fast-growing populations of cells, the vector needs to replicate alongside its host, or it will be lost from the...compatible ori that will be recognized by its host’s replication machinery. A shuttle vector designed for unrelated...experiment. Yeast shuttle vectors have these replication sequences for their yeast targets as well as ...particularly mammalian cells like neurons, may not replicate during the course of your experiment, meaning ...

...two types of replicate controls: technical and biological. In general, technical replicates can be thought... employ transfection, negative, positive, and replicate controls.  Transfection controls: empty vector...the interrogation of the independent variable. Replicate controls: technical and biological Reliable results...derived from one source. Conversely, biological replicates ARE independent and can be thought of as "reproducibility...reproducibility controls". Biological replicates are what makes up your sample size (aka your n value)"... provide more in depth information. Technical replicates An example of a technical control is the transfection...plasmid from the same aliquot/preparation. The replicate control isn’t measuring or assessing reproducibility...

...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...positives in original studies and false negatives in replications are unavoidable, but can be minimized with high...obtaining a different result after conducting a replication study. An important difference between the original... methods, QRPs, and small samples, then when replications do not come out the same as an original research...data, materials, and code Only when you try to replicate the work of other researchers (or your own from...

...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...adoption of policies that promote reproducible publishing practices. One example is the publication of peer...Wednesday we worked with the Harvard GSAS Science Policy Group to organize a Minisymposium on Reproducibility... out on the panel, irreproducibility undermines public trust in science and research. Nonetheless, I’d...difficult to publish so-called “negative” results that conflict with our original hypotheses. Looking for novelty...these positive results will be published. The publication record will then show that the tested hypothesis...

...recombination. Yeast Replicating plasmids (YRp):  These vectors contain an Autonomously Replicating Sequence (ARS...mammalian vectors, will catch you up on the core replication and resistance features of yeast vectors and ...includes but is not limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic...vectors must contain a yeast-specific origin of replication (ORI) and a means of selection in yeast cells...antibiotic selection markers.  The yeast origin of replication (Please note: This first section primarily pertains...ve also noted some features required for the replication of fission yeast, Schizosaccharomyces pombe, ...within a yeast vector determine how the plasmid is replicated and maintained within the yeast cell. These elements...

... to verify there’s no genomic integration Replicating EBNA1 episome Non-integrating Medium Medium ...creates heterogeneous iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing...Retroviruses are not considered safe for clinical applications.     Lentiviral: Lentiviral vectors infect dividing...unavoidable heterogeneity between clones. This can complicate comparisons made between samples, i.e. transgene...delivery is not considered safe for clinical applications. Excisable Cre-lox lentiviral vectors: This...is still not considered safe for therapeutic applications. piggyBac (PB) transposon: The PB transposon...express reprogramming factors for therapeutic applications. Sendai viral vectors: Sendai virus is a single...

... to test reconstitution via mRNA trans-splicing. A cis-splicing vector served as positive control (cis-ctrl... vector 2; BD, binding domain; SDS, splice donor site; SAS, splice acceptor site; pA, polyadenylation ...3’ long terminal repeats (LTR). The 3’ LTR is duplicated during lentiviral integration, producing a second...reused from Walton et al. 2024 under a CC-BY-NC-ND license. In this iteration, the size of the lentiviral...Image adapted from Yan et al. 2024 under a CC-BY license. Yan, J., et al. (2024) Improving prime editing...adapted from Serrano et al. 2023 under a CC-BY-NC-ND license.  Integrating hIBA1a promoter into AAV vectors...for microglia research and broader therapeutic applications.  Find microglia-targeting AAV plasmids here...

...your specific application. Consider also that there may be variations within an application. Take immunohistochemistry...website highlights a variation of your application, a publication successfully used it in a similar way...choosing an appropriate antibody first look at the application-specific data provided on the manufacturer’s ... a linear antigen tend to work very well for applications that denature a protein to its primary (linear...the most similar to yours. Take a look at the publications cited on the manufacturer’s website, which may...plan to. To secondary or not to secondary?  The application will also determine whether you should use an... as it will help to amplify the signal. Some applications use either direct (primary-conjugated) or indirect...

...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...of biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...secondary antibodies.   Application-specific controls Some antibody-based applications have very specific ...then moving on to controls for antibody-related applications. After reading, you will have a better understanding... be able to confirm that the testing protocols elicited the expected response and have a baseline with...statistical power of an experiment. Technical replicates are repeated measurements of the same sample ...difficult to obtain meaningful results.  Biological replicates are distinct samples that are treated identically...

... be run in duplicate or triplicate. The closeness of the data points generated by replicate values provides... the standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve... deviation of each replicate measurement divided by the mean of all the replicates multiplied by 100. ...regularly, and mix samples very well.  Use duplicates or triplicates to increase the accuracy of your experiment... critical for a number of popular scientific applications, and the quality of a standard curve can make...for an acceptable R2 but for many scientific applications users aim for an R2 above 0.95.    Additional...ideal %CV is less than 15%. You should also run replicates for all of the samples in the assay, as this ...

... recombination. In 2007, Joshua Sanes and Jeff Lichtman, both of Harvard’s Department of Molecular and...towards single-cell resolution imaging. Sanes and Lichtman endeavored to create a map of the brain, a connectome...constant inversion of the construct. Sanes and Lichtman used pronuclear injection to insert these Brainbow...table to the right); in various reports, Sanes and Lichtman have observed from 90-160 distinct colors due ...of farnesylated derivatives enables labeling of delicate axonal and dendritic processes not previously ...Between Brainbow Systems   Brainbow variants and applications beyond the mouse brain In addition to improving...improving the Brainbow system, Sanes and Lichtman also developed a complementary Flpbow construct that is ...

...Rep78/68 play a key role in AAV genome replication. Replication initiates from the AAV genome ITRs on ...contains the proteins needed for the virus to replicate,  and the transfer plasmid which contains the ...aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid (cap) gene. In wild-type...its products. Created with BioRender.com   Replication (rep) gene The rep gene encodes four proteins...proteins that are required for viral genome replication and packaging: Rep78, Rep68, Rep52, and Rep40. Rep gene...small (Rep52 and Rep40) Rep proteins. Alternative splicing generates the different C-terminal regions of ... domains, all four Rep proteins share a common helicase and ATPase domains. To function, the Rep proteins...

... DNA to silica columns, it’s bound to glass syringe filters. Normally DNA does not bind silica or glass...Regeneration of silica columns for DNA purification Starting material: PCR product or agarose gel slices Product...Before you reach for that silica spin column, stop to consider some ways to purify DNA without a kit....purification without a kit as well as one way to reuse silica columns from DNA purification kits. Kit-free alkaline...Starting material: bacterial cultures or agarose gel slices Product: plasmids or DNA fragments The plasmid ... with glass milk Starting material: agarose gel slices Product: DNA fragments You might be wondering, ...stick to the glass particles in glass milk, gel slices are first placed in a chaotropic salt sodium iodine...

...experiment is targeted amplicon sequencing. The information from targeted amplicon sequencing is highly ...have been widely adopted due to their relative simplicity and low cost. The problem with these assays is... genome for off-target sites irrespective of in silico prediction. These techniques differ in important...heterogenous populations, low sensitivity Targeted Amplicon Sequencing Provided 0.01% (variable) Sequence-...rate and the repair product sequence. Targeted amplicon sequencing doesn’t tell the whole story ...These strategies can be used in concert with in silico prediction to create a more comprehensive list ...unbiased detection isn’t as sensitive as targeted amplicon sequencing. For example, GUIDE-seq seems to have...

...targeting the pMB1 replicon on pTargetF (Jiang et al., 2015). In addition to the pMB1 replicon, pTargetF contains...PrhaB, 2) changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB... the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose inducible lambda-red... expression of the sgRNA that targets the pMB1 replicon, resulting in cutting of the plasmid. At this ... the gRNA on pCas that is specific to the pMB1 replicon of pTargetF may exhibit a more serious leaky expression...serve to interrupt genes (or gene clusters) in metabolic engineering. So far, we’ve used the MUCICAT system...when compared to the industrial strain. In our metabolic engineering case study of N-acetylglucosamine ...

...cutters. You can also sort enzymes by clicking on column heads. Clicking on a particular restriction enzyme...while the SnapGene powered map is on the right (click here to see the new map in action). Read on to learn...simple but powerful display improvements, when you click on any plasmid map, you also have the ability to...analyzer will allow you to dive into the details. Click on the “View all sequences” button to find all available...For more immediate, high-level analysis, you can click directly on the “Analyze Sequence” button to pull...Maps This is the first tab you’ll land on after clicking the “Analyze Sequence” button. The maps hosted...and a description of its function. You can also click one location in the plasmid and then a second location...

...led to their application for basic and translational research.  Basic research applications of scFvs: ...made up of glycine and serine with dispersed hydrophilic residues for increased solubility (Monnier et...blood clearance, which is useful for imaging applications; better tissue penetration which is useful for... for therapeutic and imaging applications; and reduced immunogenicity when administered in vivo due to...from blood can be a drawback for therapeutic applications where longer retention times often increase ...since it’s genetically encoded.   Translational applications of scFvs: Just like antibodies, there are many...2019).Many of the scFvs designed for oncology applications are reformatted as bispecific molecules which...

... and comment online! Want the general public (say, policymakers or community organizations) to use your...poster or talk, or submitting patent or grant applications; to informal settings like lab meetings, social...scientific manuscripts shared by the authors on a public server independent from peer-reviewed publishing...courtesy of Henry Mühlpfordt, under a CC BY-SA license. Many scientists and advocates have argued ...increasingly growing time from submission to publication. It takes many months, often more than a year...avoid many of these issues, by sharing their work publicly and immediately once the manuscript is drafted...and updated by the authors over time, providing a public record of changes, critiques or endorsements. In...

...epigenetic modifications across the genome) has been implicated in disease states, and targeting the epigenome...to specific DNA sequences. Non-editing CRISPR applications direct catalytically dead dCas9 fused to a variety...small DNA methyltransferase from the prokaryote Mollicutes spiroplasma (M. Sss1) (termed MQ1.) The Q147L...doi.org/10.1038/nbt.3199 Find plasmids from this publication at Addgene. Klann TS, Black JB, Chellappan M...doi.org/10.1038/nbt.3853 Find plasmids from this publication at Addgene.  Lei Y, Zhang X, Su J, Jeong M, ...10.1038/ncomms16026 Find plasmids from this publication at Addgene. Liu XS, Wu H, Ji X, Stelzer Y, Wu.../j.cell.2016.08.056 Find plasmids from this publication at Addgene. Maeder ML, Angstman JF, Richardson...