...search-and-replace” genome editing technique that mediates targeted insertions, deletions, and all possible...without double-strand breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime editing...editing, as it can edit near to or far from PAM sites, making it less constrained by PAM availability ...over 30 base pairs for prime editing. Since PAM sites occur on average every ~8 base pairs on either DNA... up to 45-fold) higher across different genomic sites compared to PE1 (Anzalone et al., 2019). PE3: Resolving... DNA to favor the edit Once the prime editor incorporates the edit into one strand, there’s a mismatch...additive improvements. These variants are worth testing if you want to make your prime editing as efficient...

...of most CRISPR systems and generates double strange breaks at target sites 5' of the 5'NGG3' PAM sequence... version of CAG promoter that the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of ... for more information). V2.0 This designation separates pSpCas9(BB)-2A-Puro (PX459) V2.0 from it's predecessor... has a U6 promoter followed by BbsI restriction sites into which you can clone your 20 nucleotide guide...FLAG tag at its N-terminus. This tag can be used to test for SpCas9 expression post transfection by western...

...of Brainbow, Brainbow 3.0 (Cai et al., 2013), incorporates the highly photostable fluorophores mOrange2...Tissue-specific Cre recombination in this mouse generates cells that randomly express either nuclear GFP... to describe the self-renewal of cells in the intestinal crypt, and since then has become a powerful tool... lab also developed a version of Tetbow that incorporates chemical tags instead of XFP to optimize it ...expansion and dedifferentiation of mature pigmented melanocytes." Cell stem cell 21.5 (2017): 679-693. PubMed...PMCID: PMC6245733. Snippert, Hugo J., et al. "Intestinal crypt homeostasis results from neutral competition...PMCID: PMC4077948.  Yan, Kelley S., et al. "The intestinal stem cell markers Bmi1 and Lgr5 identify two ...

...biotin-binding sites on this complex can bind to your biotinylated antibody. This creates a large meshwork...absorb most of the biotin they need in the small intestine from the microflora that reside there. Biotin ... organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier from one compound...observed that animals fed a diet of primarily egg whites would develop skin lesions and other terrible health...for easy detection. The multiple biotin binding sites found on avidin are also ideal for signal amplification...of the E.coli BPL, BirA. If you recall, BirA biotinylates only one lysine in the entire E.coli proteome...

...for seeking knowledge. The process begins with a testable hypothesis - an educated guess about how some ...of the world functions. Experiments come next, testing the correctness of the hypothesis. The results ...march in Washington, D.C., the seat of the United States government? Politics and science are intrinsically...Environmental Protection Agency, and the National Institutes of Health endanger scientists’ ability to uphold...these risks include: 1) Lost trust Perhaps the greatest risk is that the public will lose trust in scientists...scientist, Matthew Niederhuber, said, “if the march motivates more scientists to reach out to their communities...Center: The Politics of Climate Change in the United States CNBC: Kellyanne Conway: “I”m not in the job of ...

...closed states (A) and (B) but with much lower affinity than their open states (C). Figure courtesy of IPI...conformational states: bent-closed, extended-closed, and extended-open. In the two closed states, integrins...bind RGD-binding integrins at non-ligand-binding sites and do not interfere with integrin function. Overall...writer at the Institute for Protein Innovation. She writes about all things IPI, from the scientists and scientific...

...advantages over antibodies (Ahmed et al., 2012, Bates and Power., 2019): rapid blood clearance, which ...likelihood to aggregate due to their small size (Bates and Power., 2019). Their rapid clearance from blood...therapies in clinical trials or already in the clinic (Bates and Power., 2019).Many of the scFvs designed for...intracellular T cell receptor portion of the CAR facilitates signal transduction and release of cytotoxic ...2012:1–15 . https://doi.org/10.1155/2012/980250  Bates A, Power CA (2019) David vs. Goliath: The Structure... (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin...research from the Trimmer lab Check out Addgene's latest project to develop an open antibody resource ...

...the blocking-only templates will compete with the blocking/intended mutation templates resulting in cells...Ludwig-Maximilians-University in Munich and Marc Tessier-Lavigne’s lab at the Rockefeller University in ...have focused on strategies to improve overall HDR rates, we have shown recently that desired genome-editing...unwanted indels. Following this observation, we tested a strategy that was discussed in the field at the...described project while he was a Postdoc in Marc Tessier-Lavigne’s lab at the Rockefeller University in ... NYC.   Dylan Kwart is a graduate student Marc Tessier-Lavigne’s lab at the Rockefeller University. He...

...start defining keywords you’ll want to find in candidates’ resumes or CVs; “vivarium”, “weaning”, “colony...will help save time by screening out irrelevant candidates, and, more importantly, it’ll help you define...personnel salary ranges, go to online job information sites (like, indeed.com, or salary.com). Search industry...is important to ensure that you’re looking at institutes that have the means to support your work. For...work requires specialized equipment, try finding institutes that offer shared equipment that you can use ...the anxiety that comes with negotiations. This creates a self-assured discussion when it comes time to...faculty position because none of the offering institutes were able to provide the resources required for...

...the other hand, the process in eukaryotes is much more complex. Eukaryotes require a minimum of seven transcription...actually begins. Only eukaryotes and archaea, however, contain this TATA box. Most prokaryotes contain a sequence...are common in prokayotes, specifically bacteria, but have also been discovered in eukaryotes. Operons consist...binding is very different in bacteria compared to eukaryotes. In bacteria, the core RNA polymerase requires...Transcription is tightly controlled in both bacteria and eukaryotes. Promoters are controlled by various DNA regulatory...promoter also contains transcription factor binding sites, but mostly contains regulatory elements. Eukaryotic...Specific Drosophila promoter containing Gal4 binding sites Bacterial Promoters Promoters in bacteria contain...

...analyze sequence tool: Annotates features, BLASTs, aligns, digests, and translates a sequence.   NEBCutter2...restriction enzyme cut sites Webcutter 2.0: For finding restriction enzyme cut sites Open reading frame finder...sequences in a database ExPASy translation tool: Translates a DNA sequence using all six open reading frames...primer design tools, but here are a few stand-alone sites for general primer design, as well as primer design...

... viruses. Now focusing on plant biology, she coordinates the Network for the Genetic Improvement of Cowpea...agricultural plant. Her current research focuses on endophytes, bacteria, and fungi that reside within living...proteins and enzymes. These compounds will later get tested for their potential agricultural and medical use...detect glyphosate via H2O2, one of the reaction’s metabolites. One useful genetically encoded H2O2 probe is...projects progress. Subscribe to the Addgene blog for updates and future collaborations. Many thanks to all of...References 1. Martinez-Klimova, Elena et al. “Endophytes as sources of antibiotic” Biochemical Pharmacology...

...separate resistance cassettes and your gene of interest is cloned into/inactivates or completely removes...translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes.     **Dissolve in dH2O and sterile filter unless... are trying to disrupt or delete, which both inactivates the gene and acts as a marker for the mutation...months. Ampicillin breaks down especially fast and plates should be used witin 1 month for optimal efficiency... them to media hotter than about 55oC and store plates/stocks wrapped in foil if a light-sensitive antibiotic...

...mentorship is by recruiting your own peers and classmates! Your mentors don’t need to be more senior or...that there’s some correct level of intimacy that creates the magic of a safe space. No matter why, now is...roles but for almost every position, we look for candidates that have demonstrated initiative. In fact, hear...opportunity to act or take charge before others do”. Candidates who have “started something” which they can share...have a definite advantage in our process. We see candidates who have done the following types of things: ... a character count in your head.  Here are some sites that have compiled lists of grants: Science: ...

...“PCR test” has recently transitioned from jargon to non-jargon in the context of disease testing, it can...other uses. Many laypeople would use “PCR test” to mean “a test that uses a biological sample to look for...presence of a virus.” A scientist would likely mean “a test for the presence or absence of a predefined DNA ...Science to Elementary and Middle Schools   Image courtesy of Ron Mader, via Flickr, under the Creative Commons...

... scores. That player doesn’t trust his teammates; the teammates don’t trust him. This team is not going...includes: Michelle, the Senior Scientist who coordinates all kit creation and quality control efforts ...informed decision about something? Fix a problem? Test a hypothesis? Whether you officially call it a “...I’m an introvert!” is not an excuse. Every psych test I’ve ever taken has declared me an introvert, and...academic labs, uses Slack constantly. Whether your teammates are two desks away or on the other side of the...perfectly clear lines of communication, but if teammates don’t trust each other, none of that matters. ...

...way to re-create a functional virus. Indeed, by testing several vectors and packaging cell line combinations...proto-oncogenes or with human samples that have not been tested for HIV.  If not using proper technique, there’... from the LTRs) has been removed, Rev (which facilitates nuclear export) is expressed from a separate ...by your transfer vector) binds to the TRE and activates the expression of your gene. The addition of Dox...virus precipitation. My tip for this process is to test several protocols that have been described (protocol...viral capsid antigen p24, the FACS dosing method correlates viral titre to the number of cells transduced...ratio of virus to cells). Again, my tip here is to test several protocols as the set-up depends on the type...

...Surveyor assay to look for genomic edits at the BRAF sites targeted by my specific gRNAs (Figure 4). The most...the 428 bp Surveyor nuclease cleavage product indicates a gene edit. GFP gRNA-transduced Cas9-A549 cells...cleavage products. The appearance of the 428 bp band indicates edits in the BRAF gene in the BRAF-gRNA transduced... product. Figure 5: gRNA-targeted BRAF sites were edited in A549 cells. Surveyor nuclease was...those formed from control (GFP gRNA-targeted) BRAF sites (lanes 3, 4) were used as controls. Duplexes were...a gene knockout was straightforward, and is a testament to how far genome engineering has come. For comparison...Population by Limiting Dilution Lentivirus Production Websites Primer3: for Designing Primers UCSC Genome Browser...

...broth and on the agar plates. Mycoplasma colonies are easily visible on the agar plates. This method is capable... to poor lab techniques. These include reusing pipettes for multiple cell lines rather than using disposable...always use a second assay to be 100% sure. Before testing for mycoplasma, the cells should be in continuous... for low levels of contaminations to grow. For testing, the medium should not have been changed for at... (Uphoff and Drexler, 2011). Culturing on agar plates/broth is considered the ‘gold standard’ for detecting... are tightly packed with seals. Use disposable pipettes to avoid cross-contamination. Make sure you keep...

...and not exposed to CO2/dry ice. All samples were tested in duplicate. What we found Not all tubes (or ...) were permeable to CO2 at low temperatures. We tested two different types of tubes: the ones we use to...shown). What we did next: Venting the tubes We then tested whether venting the tubes prior to thaw released...What about putting it back in the freezer? We then tested whether returning the tubes to the -80°C freezer...would prevent the pH change upon thaw (Fig.4). To test this, we froze the tubes at -80°C and then moved...affects AAV function. Furthermore, another study tested AAV infectivity after a range of pH exposures. ...