We narrowed to 965 results for: REP

..., E4, and VA RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...herpes simplex virus to facilitate efficient replication. The most common production system, however, ...been developed to determine the titers of rAAV preparations, such as dot blot, enzyme-linked immunosorbent...different titers are measured. A TCID50 measurement reports an infectious titer, meaning the concentration ...targeting the viral genome or inverted terminal repeats (ITR). You should always have a high-quality plasmid...overestimate of transducible units in the virus preparation.  rAAVs  Now your AAV virions are ready to store...

...along with a reporter, such as a fluorescence gene or a resistance gene. These reporters allow you to ...multicistronic element(s), or you could replace one of the reporter genes above with your gene or genes of... using IRES elements. Further, scientists have reported lower expression of the downstream cistron due...eukaryotic cells, and, although some scientists report close to 100% cleavage with some of these peptides...that already has the multicistronic element and reporter cloned in. In these plasmids you would simply ... 2A element (depending on the placement of the reporter gene). Addgene's collection offers a variety of... are designed to have one or more of the genes replaced.  Additionally, 2A peptides could be PCR-cloned...

...that can be repaired by either non-homologous end joining (NHEJ) or homology directed repair (HDR). This...photoactivatable repressor (dCas9). The activity of all variants was reversible and repeatable. Since these... spatiotemporal control, reversibility, and repeatability. The system developed by the Sata lab is composed...could be spatially controlled in a reversible and repeatable manner. With their light-activated CRISPR/Cas9...The system was also shown to be reversible and repeatable when blue light (450nm) was cycled on-off-on....system, Ye’s lab could edit a tdTomato fluorescent reporter gene in mice using Minicircle DNA vectors encoding...seen in the figure below. Next, by using a dual reporter fluorescence assay, Hemphill et al. demonstrated...

...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...mutation than restriction enzyme based cloning. DNA replication by PCR has error rates that range from roughly...

... countries, into our repository, while expanding global access to our repository through our distributor...At 3500 plasmids, this represents the largest single deposit in our repository and was a three–year project...14 deposits, that became available through our repository in 2023.  In June, we announced a new partnership... launch our Antibody Data Hub, an open-access repository for antibody validation data. We’ll continue ...expanding international access to the Addgene repository, partially through work with our distributors...we are now able to ship rabies plasmids in our repository to international requestors.    I would be remiss... used by our requestors, and supported by our repository. You can enjoy these stories, and other 20th ...

...Sanger sequencing to confirm an insert that is repetitive or otherwise hard to sequence using NGS or long-read...such as G-C rich areas, secondary structures, or repetitive regions. Short-read sequencing can also be time-consuming...time-consuming, depending on the library prep and instrumentation used.  Here at Addgene, we sequence ...sequencing or Sanger sequencing, either because of repetitive regions or for higher accuracy of a particular...This method is great for resolving complex and repetitive sequences, but can be costly, low-throughput,...Nanopore sequencing to QC plasmids that are highly repetitive, such as those containing cassettes of the same...the reads, which boosts ‌accuracy, especially in repeated regions. If this doesn’t resolve the assembly ...

...storage, viral preps should be divided into single-use aliquots, and stored at -80°C. Some reports suggest rapidly... time consuming, difficult, and lacking in reproducibility. Read on for some tips for getting the most...Obtain a vial of the desired cell line from a reputable distributor such as the American Type Culture ...you may want to consider concentrating your viral prep. Storage can also play a role in the success of ..., it is best to avoid freeze-thawing lentiviral preps unless absolutely necessary. Many labs try to use...this blog post for tips on titering your lentiviral prep!) and infect. While this approach is a great start...transducing with a range of dilutions of your lentiviral prep. Hopefully, by using more concentrated dilutions...

...sites if in double stranded DNA are repaired by base excision repair, but can lead to mutations in single...noticed that there are many options to elute your DNA prep. You might see protocols that recommend eluting ...single stranded DNA undergoing replication.   DNase on the other hand can digest and degrade the double ... buffer for preserving the stability of your preparation for a long time. Tris buffer controls the pH,... if the priority is to directly use your DNA preparation in any downstream application involving enzymatic...buffer is that it allows better control of the pH preparation stabilizing the plasmid, and still allowing most...at -20°C or even at -80°C, in this case your preparation can be eluted in water or in your buffer of preference...

...confirm the identity and serotype of our AAV preparations. We recently published a description of the ... For labs that produce a small number of AAV preparations this level of quality control is sufficient ...facilities producing a large number of viral preparations in parallel, there is a very real risk of mixing...AAVs and the TA-based ligation and tagmentation preparation approaches described previously require a preliminary...effective substrates for tagmentation-based library preparation procedures. This finding eliminates the double-stranding...simplified approach, we sequenced over 100 AAV vector preparations and showed that VGS can quickly and reliably... script that can confirm the serotype of AAV preparations.  Using viral genome sequencing to determine...

...platform for scientists using Addgene AAV viral preps to easily find and share useful technical data. ...tools. This word cloud (using wordart.com) represents contributed data on brain injection site from...15 minutes or less if you have all of your data prepared. All of these questions relate to factors that...that you should have already recorded as good reproducible and record-keeping practice. In fact, when you...did you use? For animal subjects it is helpful to report the genetic background and catalog number or RRID...exactly which strain you used so if they wish to replicate your experiment, they know exactly where to get...take negative data, which is equally important to report. We recently published a paper in Frontiers in ...

...ACE) in real-time. The reporter constitutively expresses eGFP to indicate reporter abundance, and mCherry...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These articles...and N177F mutations). Its excitation maximum is reported as 603 nm, while the emission maximum is 675 nm... ACE system: A versatile in vivo fluorescent reporter for rapid quantification of base-editing and Cas9...editing tools, the Reuben Harris lab has deposited a reporter plasmid that allows for rapid fluorescence-based... out a frame-shift mutation within the mCherry reporter. Quantification of editing efficiencies simply...containing rat APOBEC1. Finally, they show that the ACE reporter can also quantify Cas9 cleavage, thus exhibiting...

...can treat the underrepresented individuals too nicely. When the work of the underrepresented individual ... the academic research environment is setup to prepare trainees for further work in academia. In fact ...Increase the number of science masters programs to prepare scientists for positions outside of academia Require...(the current median postdoc starting salary as reported by Paula Stephan is $40,000) Increase Knowledge... Duchin provided a concrete example of how underrepresented individuals are discluded/not treated equitably...well-meaning mentors often fail to judge the work of underrepresented groups under their mentorship with the same...third party, it can come as a shock to the underrepresented individual. In an attempt to be nice, the ...

...while meditating right before bed will help you prepare for sleep. At Addgene, our mindfulness group is...more about career paths and things you can do to prepare. Adapt to WFH while parenting Keep your kids...career exploration site Inventing Heron has a radio reporter challenge, and code.org has coding tutorials for... for all ages. Organize your data and prepare publications or your dissertation Organize data that you...write introductions to potential publications or prepare a review article. These are both options that can... your defense less stressful. Think about reproducibility best practices Support your future experiments...experiments and other researchers by implementing reproducibility best practices in your research. For a place...

... are replication incompetent and cannot readily be passed on to subsequent cells via replication like ...usually spread unidirectionally, there are some reports of bi-directionality. For predominately anterograde...cleft is usually severely impaired, and only few reports exist about dye molecules that are transported ...infection to the central nervous system where it replicates, spreads, causes neurotoxicity, and is lethal...HSV) also migrates through neurons where it can replicate and by doing so spread across several synaptic...cells are labeled overtime due to the continued replication of the virus. While this can be useful, if too...single-stranded DNA molecule and AAV vectors are replication deficient (i.e. they cannot easily spread between...

...enough information to replicate your experiments, making research more reproducible and your results more...scientific communities. The ARRIVE guidelines for the reporting of laboratory animals during in vivo research ...research and the CARE guidelines for case reports are two sets of guidelines frequently used by editors. Providing...communities aim to significantly improve the reproducibility and reliability of science publications. Peer...s name and affiliations, and the entire referee report are permanently attached to the published article...and readers are able to comment on the referee reports. If the reviewers suggest that the manuscript requires...article, while the first version and the referee reports remain available online. Open-peer reviewing is...

...consider using a different antibody.  Prepare a positive control Prepare and label a sample that expresses... and secondary antibody labeling and imaging, repeating for each target. Alternatively, you can perform...paraformaldehyde-fixed cell samples, or other preparations. Sequential IF For a sequential IF experiment...wash, add secondary, image again, inactivate, and repeat these cycles for all your targets. Of course, you... ICC validated antibodies from Addgene), or be prepared to run some extra controls and validate the antibody...then label that with fluorescently-conjugated streptavidin to get more fluorophores per antibody, or conjugate...the offending red-green image, go to Image>Color>Replace Red with Magenta. Or you can use Color>Split Channels...

...Unreliable antibodies cause research that cannot be reproduced and projects that cannot be completed. A 2015...data that they are a long-term solution to reproducibility and cost-effectiveness for broad and large ...criterion that includes accuracy, strength, and reproducibility. We test the antibodies on human and mouse ...comparison with two atlases enables us to see the reproducibility and accuracy while screening. We also compare...exposure time closer to the negative control. Reproducibility  In order to attain non-bias assessment, multiple...and exposure times. To test the antibody’s reproducibility, each antibody is independently screened twice...References and resources References Baker M. Reproducibility crisis: Blame it on the antibodies. Nature....

...format) At least two replicates of your experiment* Functional data, if the report describes a functional...searching the Data Hub's curated collection of reports (which you can filter by serotype, species, or ... significant time and money. For instance, this report shows that anterograde, monosynaptic connections...our AAV Data Hub Challenge recording to see two reports presented by the submitting scientists, learn more...injection site as well as the target region, if the report describes a retrograde serotype An abstract that...experiment clearly and concisely* *required for a DOI. Reports that meet our minimum standards (* above) will ...submissions will receive a 20% off coupon for a viral prep. And if you've used the Data Hub to help select ...

... is a transposon? DNA transposons are mobile, repetitive genetic elements that are found in both prokaryotic...This is similar to how retroviruses, like HIV, replicate. Class I TEs do not encode a transposase enzyme...enzyme.  Class I TEs are considered replicative since every time they jump they make a copy of themselves...types of Class 1 TEs: those with long terminal repeats (LTRs) and those without (non-LTR TEs). LTR retrotransposons...retroviruses both in their structure and mechanism of replication. They contain two genes: gag and pol. The pol...they move. Most Class II transposons have a non-replicative “cut and paste” mechanism of transposition: they...transposase enzyme binds the transposon’s long terminal repeats (LTRs), induces a double-stranded break, and excises...

... Schematic representations of the indicated unbiased genotyping options. Figure reproduced from Tsai and...Verifying these connections can help alleviate the reproducibility crisis in biology. It is key to address these...valuable step toward enhancing confidence and reproducibility of a study’s findings. Table 1: Biased Genotyping...Low frequency off-target effects may generate irreproducible data in a research setting, but these events...off-target sites including both the edit rate and the repair product sequence. Targeted amplicon sequencing ...direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (BLESS) (Crosetto... unbiased methods, high background BLESS Not reported Cell-based (in vitro) Can be used on tissue from...