...any nascent technology, researchers are having trouble getting NgAgo genome editing up and running in ...get a better idea of how many people are having trouble, to determine if there have been any early successes... users likely need more time to optimize and troubleshoot the system. Site Directed Mutagenesis by PCR... with Enhancers and Epigenomics 5 Tips for Troubleshooting Viral Transductions Single Base Editing with...

...can easily spot the colors and text. Tip 2: Use electronic to-do lists to access your tasks on the go Never...could easily lend one to another labmate? Though electronic lab notebooks (ELNs) have gained traction with...to borrow most frequently. Copy these into an electronic file sharing tool, such as Google Drive or Dropbox...documents from anywhere, but also allow you to control who can edit the documents and track the revision...

...serotype-specific differences in tissue and/or cell type tropism. Introducing changes and/or short peptides sequences ...variable loops is one method for changing a capsid’s tropism.  cap also encodes the Assembly-Activating Protein...References: Naso MF, Tomkowicz B, Perry WL III, Strohl WR (2017) Adeno-Associated Virus (AAV) as a Vector...

...          Special Thanks Trophies and engraving services from Hit Trophy Additional Resources on the...positively affect scientific research. As Maria Castro of University of Michigan says, “Addgene has transformed...distributed more than 100 times. We hope these desktop trophies will serve as badges of honor for our powerhouse...

... organs located deep in the body, like the gastrointestinal tract. Require Administration of Substrate...pharmacokinetics and biodistribution therefore introduce additional confounding factors when attempting...reporter genes for in vivo imaging Following in vitro characterization, Bourdeau et al performed a proof-of-concept...coli aided in ultrasound imaging of the mouse gastrointestinal tract. The E. coli strain Nissle 197 (ECN)...

...range from researching an area, to writing an introduction that gives a basic overview of kinematics in... to students, like how to pour a gel [for] electrophoresis or doing a dissection video. I did that in ...traveling with the sales team to meet faculty and introducing the kits to clients who might initially be doubtful...themselves. “You open up the box and there’s a gel electrophoresis tray in there. Or the dissection specimens....

... eSpCas9 and SpCas9-HF1. By mining the treasure trove of naturally occurring Cas9 diversity via the Integral... Table 1. Comparing SpCas9 and GeoCas9. *For in vitro Cas9 RNP activity or longevity. GeoCas9 is active...Besides being able to take some heat, GeoCas9 can introduce genomic edits in mammalian cells. Harrington et...GeoCas9’s thermostability could also be used with in vitro molecular bio applications that require high temperature...

...it is non-invasive and offers a high degree of control over time and space. Zebrafish have become a popular...are not native to vertebrates and are commonly introduced in zebrafish through the UAS/Gal4 system for ...had previously been a mystery, but through the control offered by the optogenetics toolbox and the versatility..., Filippo Del Bene, and Herwig Baier. "Optical control of zebrafish behavior with halorhodopsin." Proceedings...

...Cas9 (dCas9) or a Cas9 nickase (nCas9) to avoid introducing double-strand breaks. The only things base editors..., the team noticed that this editor sometimes introduced C → G edits at certain positions within the editing...during cytosine-to-uracil editing, and instead introduced its opposite, a uracil DNA N-glycosylase (UNG...end joining (NHEJ). To mitigate this, the team introduced the enzyme HMCES, either as a fusion construct...ever. References and Resources Glossary We introduced a lot of terminology in this post! Here’s a quick...

...group optimized their initial system by using a stronger promoter and different biochemical secondary energy...cell free system allows for a relatively simple, controlled “one-pot” reaction capable of reproducing useful...codons, splice acceptor and donor sites, and introducing premature stop codons.    New from the viral... and 5HT sensors New AAV tools to selectively control parvalbumin interneurons from the Dimidschstein...

...translated into proteins, or, in the case of retroviruses, reverse transcribe their gRNA into DNA, which...is. Thankfully, through the use of engineering controls — changes made to the virus that eliminate or ...viral vectors to reproduce only in experimentally controlled conditions, making them far safer to work with...they’re derived from: AAVs, lentivirus, gamma retrovirus, and adenovirus. And now that you know what viral...

...epitope tags? Epitope tags are short peptides introduced at the N or C terminus of a protein that are ...hundred amino acids, depending on the tag, and are introduced via knock-in at a genomic locus or more commonly...should be used with caution or not at all. The introduction of a tag at the N or C terminus of a protein...  Susanna Stroik...

...chemically or electro-competent E. coli - excess salt causes arcing during electroporation which would ...sequenced for potential positive clones. Once you introduce your 3’-end “A” overhang insert, the magic of ...would cause the electroporation to fail). When incubating at room temperature, it is not recommended that...

... promoters are upstream or 5’ of the gene they control. Promoters can be organism or tissue type-specific...different levels of expression. For example, CAG is a strong promoter that drives very high, ubiquitous expression...recombinase, are used to spatially and temporally control transgene expression. Lox sites are directional...availability of Cre, lox sites lets researchers control the spatial and temporal activation of a transgene...

...increased yields in adenoviral (Coroadinha, 2006) and retroviral production systems (Shen, 2011). To our knowledge...also improved yields of the AAV2 derivative, rAAV2-retro, but had no effect on AAV5 yields. We further show...preparation. Through sugar detection assays, in vitro transduction, and silver stain approaches we show...Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability." Biotechnology...

...PubMed PMID:29109157. Nanobody toolkit to monitor retrograde transport at the cell surface Article contributed...developed a set of functionalized nanobodies to study retrograde transport from the cell surface. Their toolkit...treated with biotin–phenol and H2O2, APEX2 labels electron-rich amino acid side chains (e.g. Tyrosine) with...biotin-labeled proteins can them be analyzed by mass spectrometry allowing a researcher to specifically identify...

...gene-editing protein - a DNA-cleaving Argonaute from Natronobacterium gregoryi, or NgAgo for short. Addgene has ..., showing that this protein could cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately...RNA guides must be expressed from a plasmid or in vitro transcribed, and changes in gRNA secondary structure... Han. “DNA-guided genome editing using the Natronobacterium gregoryi Argonaute.” Nat Biotechnol. Epub ...

... 10. Plasmids 101: Control Plasmids Need help thinking about the types of controls you’ll need for your...provides researchers with many different ways to control gene expression. Get the full scoop from Addgenie...Addgenie Chari Cortez guides us through some common controls and why you might need them. Happy Reading!   ...

...The gRNAs targeting EGFP can be used as positive controls for gene editing or gene activation, while the...What you don't need to do is spend your time troubleshooting reagent production, because that's a distraction...from your primary goal. Getting these quality-controlled reagents from Addgene can greatly expedite a ...Research: A Primer for Beginners 5 Tips for Troubleshooting Viral Transductions Additional Resources on...

...pairs from intergenic regions, intronic regions, and a pericentromeric region. All pairs were easily visualized...CRISPRainbow. Both tools were targeted to the FBN3 intronic repeat which contains 22 copies of the gRNA target...locations of the subtelomeric region of the pericentromeric region (PR1), the p-arm (T1), and the q-arm...