...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...

...create a break that can be repaired by homologous recombination using a repair template. These templates... is to inactivate, or "knockout", the gene by replacing it or disrupting it with an piece of DNA designed... Specially constructed plasmids can be used to replace genes in yeast, mice, or Drosophila through homologous...Homologous recombination is a mechanism to accurately repair harmful double stranded breaks, in which nucleotide...targeting takes advantage of this natural process to replace a targeted genetic locus with homologous sequence...gene will be removed from the chromosome and and replaced by the resistance gene.The gene is thus disrupted...normally because one of its exons has simply been replaced with the same sequence flanked by LoxP sites while...

...double-strand break in the target DNA, which, when repaired through error-prone non-homologous end joining...target gene. CRISPR can also be used to activate or repress target genes without permanently modifying the ...libraries have been created to knock out, activate or repress target genes by combining a gRNA library with the...CRISPR libraries for gene knockout, activation, repression, and barcoding.  3) Are you trying to target...If you've obtained a ready-to-use lentiviral preparation from Addgene, you can skip the steps above! Cells...requires the use of next-generation sequencing. Representation - Most libraries contain 3-6 gRNAs per target... gRNA within the population is key. Loss of representation due to enrichment or depletion of specific ...

... deviation of each replicate measurement divided by the mean of all the replicates multiplied by 100. ...step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for each... could affect the measurements. You'll need to prepare the standard, standard dilutions, and unknown sample...components can affect the final readout. When preparing a standard curve, change pipette tips and mix ...The closeness of the data points generated by replicate values provides useful information about the accuracy...every time the assay is run, ideally with freshly prepared buffer. This is because standard curves vary depending...standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve’s coefficient...

...produce complex fluorescent reporter constructs. These fluorescent reporters can be used to detect structure...Tubulin Repeatable, directional cloning   The principle behind MXS-chaining is repeatable, restriction-enzyme...protein sequences to develop diverse fluorescent reporters is a useful application of high-throughput cloning...ligation product, which is the basis for the repeatability of the method. Note that the restriction sites...for the modular assembly of highly similar or repetitive sequences. Intermediate chaining products (called...For example, because CpG dinucleotides are underrepresented in most vertebrate genomes (Josse et al., ...genes from organisms with high CpG dinucleotide representation. The specific restriction enzymes used in various...

...combination with that reporter, you can infer its presence through detecting the reporter's fluorescence. Another...labeling proteins is to fuse them with a fluorescent reporter, such as GFP or mCherry, through genetic modification...combination of antibody-fluorophores, fluorescent reporters, and/or dye stains used in your flow cytometry...mentioned previously, you can also use fluorescent reporters, such as GFP, and fluorophore-conjugated antibodies...compare the excitation/emission spectra of your reporter with the ones from your fluorophore labels and...encodes for eGFP, which serves as a transduction reporter. Note that eGFP is not fused to CD45, but is separately...for having a separately expressed transduction reporter is that not every transduced cell will necessarily...

...highlight how to prepare and things to keep an eye out for during training. Preparation before bench work...the training. In this blog we will review the preparation and planning you can do to build a bench mentor-mentee..., or a graduate student. This is the person you report or talk to on a daily basis who will help you get...In research there is a saying that if the sample prep is poor, then the data will be poor. The same is... training in the lab! If you don’t do proper preparation and pay attention during training, then you won... you – so do your best to show up as your most prepared, can-do self and make the most of it! So, what... schedule, be respectful of theirs! Show up prepared and take training seriously, as reviewed above...

...Description Origin of Replication (ORI) DNA sequence which allows initiation of replication within a plasmid...plasmids are small circular pieces of DNA that replicate independently from the host's chromosomal DNA....virulence. All natural plasmids contain an origin of replication (which controls the host range and copy number...Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease... plasmids and the ability of plasmids to self-replicate within a cell make them attractive tools for the...plasmid by recruiting replication machinery proteins Antibiotic Resistance Gene Allows for selection of plasmid-containing...expression plasmids, gene knock-down plasmids, reporter plasmids, viral plasmids, and genome engineering...

...AAV Donor Vectors. Cell Reports 20:750–756 . https://doi.org/10.1016/j.celrep.2017.06.064  Carvalho LS...after though, three groups independently tried reproducing these results and determined that even when larger...template for CRISPR-mediated homology directed repairs (HDR). Split AAV vector approaches Split vector...(62%) vector. 4. Sequential homology directed repairs Overview: The sequential homologous recombination...templates using two sequential homology directed repairs (HDR) with CRISPR/Cas9. Donor A contains: 400bp...approach is dependent on double-strand DNA break repair, if non-homologous end joining (NHEJ) disrupts ...Mediated via a DNA-PKcs-independent, Rad51C-dependent Repair Pathway. Molecular Therapy 21:2205–2216 . https...

...the new plasmids, antibodies, and viral preps in our repository through our Hot Plasmids articles. Here's...this proof-of-concept study) are captured on streptavidin-coated magnetic beads through an assembly of... 10 minutes of glutaraldehyde crosslinking: representative micrograph (C), particle orientation distribution...RvLEAMshort to the sample before plunge freezing represents a convenient, effective approach to protecting...Integrase Gene Editing (PASSIGE), their previously-reported technique for precisely integrating large DNA ... & Liu, D. R. (2022). Programmable deletion, replacement, integration and inversion of large DNA sequences... off. Plus, DNA methylation is a long-lasting repressive marker, allowing the team to develop self-silencing...

...shared data, on our values of transparency, reproducibility, and community-building. The transparency begins...to better plan your experiments. To promote reproducibility, Addgene will have unique Research Resource...that recombinant antibodies are excellent for reproducibility because they do not share hybridomas’ genomic...drift or limited material from polyclonal animal preparations (Bradbury et al., 2015). All of Addgene’s antibodies...infrastructure, from our educational materials to our repository of open samples. We are, and have always been...2016) RRIDs: A Simple Step toward Improving Reproducibility through Rigor and Transparency of Experimental....2016.04.030    Bradbury A, Plückthun A (2015) Reproducibility: Standardize antibodies used in research. Nature...

...channel. And with no more ado, the questions!  AAV Prep and Technique Q1: Is there any way to deliver a ...you think it is possible to make an efficient AAV prep for a 4.2 kB insert with the CMV promoter using ...We have found this method to be reliable and reproducible. However, we do not use amplicons in ITR as ... in sample treatment across methods can affect reported titer by either method so to ensure equal animal...AAV8. Very recently an engineered AAV5 capsid was reported for specific and potent liver transduction. While...concentrated to ~1x1014 vg/ml.. As evident in our repository, this application is an outlier for PHP.eB (1x1011...enhanced CNS tropism to other serotypes, but reproducible success remains elusive.  There has also been...

... ready-made AAV and Lentiviral preps. This new Viral Service represents Addgene’s largest new initiative...16 labs currently sharing viral preps) through the Addgene repository. Without these scientists and the...institutions to distribute viral preps derived from plasmids in the repository. The Outreach Team has been ...provide faster access to a larger number of viral preps. Visit our Sponsors and Supporters Page Thank you...proud to be adding viral vector services to our repertoire. Additional Resources on the Addgene Blog ...

...use of both when making plasmids. Use repositories Repositories such as Addgene help share materials quickly...feels like an eternity of cloning, waiting, and repeating before you can finally dive deep into generating...everything correctly the first time – no mistakes, no repeats! How long does it take in total? While much harder...resources, and, on the rare occasion things go wrong, replace them for free. Plasmids ship out 2-3 days after...in a 96-well (or 384-well) plate! How much do repositories help?   Designing precise calculations for any...clear that community engagement with and use of repositories like Addgene provides large-scale savings. How...find plasmids Search for a plasmid in Addgene’s repository ...

...as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter lines for more sophisticated experimental...a UAS-driven luciferase reporter construct. In the absence of GFP, no reporter output was observed in ... and a reporter construct into the mouse retina. GFP expression robustly activated the reporter gene TdTomato...GFP. They then tested T-DDOGs with two mouse GFP reporter lines; again, TdTomato was seen only in cells ...tested in retinal electroporation studies, DsRed reporter expression was induced in ~76% of GFP+ cells, ...

...particles and the virus particles produced could not replicate. However, Mother Nature found a way to re-create...packaging plasmid forming a vector that could create replication competent virus. Even though this system was ...still a potential risk for the generation of replication competent lentiviruses (RCL) especially if you...factors in HIV positive cells could make the virus replication competent. The 3rd generation of lentiviral...transgenes (>8 kb) Potential for generation of replication-competent lentivirus (RCL) Efficient gene transfer...transfer Potential for insertional mutagenesis: Replication-incompetent lentiviruses with human tropism can.... The addition of Dox in the culture will then repress your gene of interest. This system is efficient...