... digital PCR is a sensitive assay, so cross contamination into a no template control (NTC) well is common...reduce aerosols that could potentially lead to contamination. Optimize your PCR For a good starting place...place on what PCR parameters to use, see Lock’s seminal paper on AAV titration (Lock et al., 2014). If you...tool for titration and integrity evaluation of recombinant adeno-associated viral vectors." Human Gene Therapy...PMID: 29548643. Lock, Martin, et al. "Absolute determination of single-stranded and self-complementary adeno-associated... PubMed Central PMCID: PMC5915492. Witte, Anna Kristina, et al. "A systematic investigation of parameters...

...alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek et al., 2012... mammalian cells by expressing dCas9 with an N-terminal 3xFLAG tag (3xFLAG-dCas9) and a gRNA targeting...this form, the CRISPR complex, consisting of a recombinant dCas9 protein and synthetic gRNA, is incubated...DUX4, a transcriptional regulator that when inappropriately expressed can cause facioscapulohumeral muscular...non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing." PLoS One 10.4 ...chromatin by in vitro enCh IP technology using recombinant CRISPR ribonucleoproteins." Genes to Cells 21.4...

...gene-transfer tools in the study of the brain and spinal cord, which together are known as the central nervous...transduction of both the CNS and PNS.That is until the Gradinaru Lab at Caltech stepped up to the challenge. Find...is the CREATE method?  CREATE stands for Cre Recombinase-based AAV Targeted Evolution. In this technique...short DNA sequences that contain all possible combinations into a region of the cap gene. In this special... one RGB AAV and red, green, or blue are the predominate colors expressed (figure 4c).   AAV Vector...therapy treatments for neurological diseases). The Gradinaru Lab has a lot going on. Here are the key takeaways...

...was over 300 years ago. Teaching by example No examination of the state of science would be complete without...story has its own Franklin in Doug Prasher, who originally isolated the GFP gene but was not included amongst...spends some time on Addgene’s role in the rapid dissemination of these technologies, proposing that “Addgene...undermining of the scientific process and the marginalization of science, particularly public health and ... observations and suggestions are not so much inaccurate as already somewhat dated. Zimmer could not have...continue to be a “critical consequence of ...the marginalization of science,” we know now in the summer of 2020...

... input and polling, Blugene's new virus friend finally has a name! We thank you for your participation...* Aavery! Welcome to Addgene, Aavery!       Original post: In December, we introduced you to Blugene...names and we’ve recently chosen four names as finalists. Now, we’re asking you to help us make the decision...meaningful names…. Watch the video to find out the final four! (Note: For cases where the same name was submitted...submitted by more than one person, we selected the finalist based on the first time of submission.)     ...

...growing amount of interdisciplinary research while at the same time increasing interdisciplinary citations in...continue to rely on printed journal articles as the dominant tool for sharing ideas. This leads to everyday... solutions to complex challenges. As a cross-disciplinary researcher, the challenge of keeping up on studies...interactive chart where you can see the yearly interdisciplinarity index of research for single or multiple ...2009): 858-862. 2. Van Noorden, Richard. "Interdisciplinary research by the numbers." Nature 525.7569 ...

... of these CRISPR systems. The original pCas/pTargetF system Our original pCas/pTargetF system consists...Originally published Apr 13, 2021 and last updated Aug 12, 2021. This post was contributed by guest bloggers... to yield pEcCas. In pTargetF, we replaced the original 20 bp specific sequences on pTargetF with the ...of the pEcCas/pEcgRNA system Compared with the original system, the pEcCas/pEcgRNA is superior in several.... The pDonor plasmid pRE57-Ter with multiple terminators can serve to interrupt genes (or gene clusters...

...cytosine or adenosine deaminases to facilitate a single base pair edit. (Cytosine deaminases edit C-to-T and...and adenosine deaminases edit A-to-G.) In short, the impaired Cas9 localizes the deaminase to where the...This post was originally written by Joel McDade and significantly updated in 2022 by Susanna Stroik.  ...choose this system.  Fig. 1: Cytidine deamination takes place on the free strand of DNA and converts...

... and decide whether you will create an N- or C-terminal fusion based on your experimental needs. Equally...have reported that FP cysteine residues form inappropriate disulfide bonds in the secretory pathway (Figure...localization. For example, the formation of inappropriate inter-chain disulfide bonds between FPs blocks...and blue variants that can be used in various combinations. For example, with standard fluorescent microscope...can label multiple proteins of interest with a combination of green and blue or cyan and yellow moxFPs. ...postdoctoral fellow at the University of North Carolina at Chapel Hill. Under the co-mentorship Blossom...

...  The Addgenies will choose up to four finalists, and the final winner will be determined by popular vote...cloistered consideration, and with much debate, we have finally narrowed it down to three choices.   Fig...unveil the winning name on Feb. 24. Stay tuned!    Original Blugene and Aavery would like your attention, ...Additionally, anyone whose submission is included in the final four will receive some rockin’ newly designed Addgene...

... 37°C heat block, LED blue light illuminator or UV light illuminator. Approach: Highly similar to the ...Cas12a activity gets activated. This unleashes indiscriminate ssDNA cleavage activity which cleaves the supplied...extracted from a sample and subject to isothermal recombinase polymerase amplification (RPA) using a commercially...amplification is done at one constant temperature, eliminating the need for thermocyclers. This is followed ...contains SSB, strand displacement DNA polymerase, recombinase, and the ssDNA-FQ reporters that exhibit fluorescence...

...-open. In the two closed states, integrins are inactive, with low affinity for ligands. When integrins...toolset IPI’s anti-integrin toolset comprises 26 recombinant antibodies (rAbs) either produced de novo using...into two groups. Seven are reconstructions of the original hybridomas as new reproducible rat or mouse rAbs.... Based on the established applications of the original hybridoma monoclonals, we anticipate that our ...other RGD-binding integrins, individually and in combination, showing that among the three αV integrins expressed...an expanding collection of readily accessible recombinant antibodies and plasmids. To encourage collaboration...

...protrusions and overlaps (arrows) after blue light illumination. Scale bar 25 µm. Images adapted from Mahlandt... 10, 30, 50, and 100 times the activity of the original, generated using directed evolution (Tei et al... can hydrolyze phosphatidylcholine (PC), the predominant membrane lipid, to phosphatidic acid (PA) or ...GFP-PASS (green). After 30 minutes of blue light illumination, substantial PA accumulates in the plasma membranes...other enzymes. Conclusion We hope this post has illuminated some of the many tools available for controlling..., & Hehnly, H. (2023). Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right...

...Nobody EVER complains that a seminar was too easy to understand. Make your seminar accessible. Let the experts... at [email protected]." You can find his original blog post below! --Ed.  Eight years ago, I decided...proper consideration. 3. Present an easy to follow seminar See item 2. Attendees will include grad students.... This isn't the time to go into detail. Finally, state how successfully completing the research...applying for It's not about you and how you will finally be granted the faculty position you have craved...

...short (1 kb) homology arms underwent homologous recombination at a frequency three orders of magnitude higher...strand invasion, a key step in the homologous recombination pathway. The Waldman lab at the Georgetown University...for the generation of knockins via homologous recombination with a homologous repair template. It is this...endogenous epitope tags, or the introduction of dominant mutations of a gene into the endogenous allele... mechanisms and consequences of cohesin gene inactivation in human cancer.     References 1. Russell, ...generating human somatic cell gene knockouts using recombinant adeno‐associated viruses." Nucleic acids research...

...1977, Sanger sequencing, also known as chain-terminating or dideoxy sequencing, was a major breakthrough... of all possible lengths because extension is terminated upon the incorporation of a ddNTP. Researchers...gels are classic techniques that allowed for many seminal scientific discoveries (5, 8). While newer techniques...techniques. For example, restriction mapping led to recombinant DNA technologies that are now commonplace in ... Alan R. Coulson. "DNA sequencing with chain-terminating inhibitors." Proceedings of the National Academy...al. "Large-scale and automated DNA sequence determination." Science 254.5028 (1991): 59-67. PubMed PMID...

...transferred to a membrane, stained with antibodies, and finally visualized.   This process allows you to detect...typically do not transfer as well using this method.  Finally, commercially available dry blotting systems efficiently...materials and methods sections of those publications.  Finally, always verify that the chosen antibody works in... these cases, the western should be repeated.  Finally, while westerns are a useful way to gauge protein...phosphorylation, acetylation, methylation and ubiquitination. For example, to test if a protein is phosphorylated...and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112:195–...

...the billions of dollars (Freedman et al 2015). Finally, as Pamela Hines of AAAS points out on the panel...enterprise towards greater reproducibility. Cause 1: Inability to repeat previously performed experiments Researchers...so-called “negative” results that conflict with our original hypotheses. Looking for novelty in our stories...similar subjects in a single, searchable space. Finally there are new publishing outlets like F1000, PeerJ... presents only the results that align with the original hypothesis. Other practices that fall into this...researcher would get in-principle acceptance of the final manuscript regardless of whether the ultimate results...

...Gautier et al., 2008). Although based on the same original enzyme, SNAP-tag and CLIP-tag show good specificity...(Erdmann et al., 2019). Check out our recent Illuminating Choices blog post for more discussion about ...Allgeyer, E. S., Erdmann, R. S., Wood Baguley, S., Sirinakis, G., Schepartz, A., Baddeley, D., Toomre, D. K... R. Y. (1998). Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells. Science,...Encell, L. P., McDougall, M. G., Hartzell, D. D., Karassina, N., Zimprich, C., Wood, M. G., Learish, R., Ohana...from Addgene Better Dyeing Through Chemistry Illuminating Choices: A Guide to Selecting Fluorescent Dyes...

...you see that there is high RNA expression in the retina and in parts of the brain, but almost none in the..., you will see a strong antibody signal in the retinal sample, but little to no signal in the liver sample...CRISPR/Cas9 to create mutations that essentially eliminate expression of the target. Removing (or reducing...interest in your sample, should lead to reduced or eliminated antibody signal. If you see a similar antibody...non-mutant line but no signal in the mutant line! Recombinant Strategies This approach is essentially the opposite...ready-to-use antibodies from Addgene Designing a recombinant validation experiment? Find plasmids here!  ...