...the RNases on your hands can degrade RNA. Keep a clean work area, which may include spraying your bench...Alternatively, glassware can be baked at 180°C for at least 4 hours.   If your final RNA sample(s) are resuspended...cell lysis.  In the late 1970’s, Chirgwin and colleagues showed that a strong protein denaturant, guanidinium...mindful of your extractions- if you need really clean RNA, it’s important to make sure that when extracting...effect of ammonium sulfate on the activity of ribonuclease A. Biochimica et Biophysica Acta (BBA) - Enzymology...active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299. https://doi.org/... Leah Schwiesow...

...macroautophagy of the cytosol leads to accumulation of Pho8Δ60 in the vacuole, where it is cleaved to generate an active... molecular weight shift of Pho8 from its uncleaved to cleaved form is the final read out of this assay...cytosol, to the higher pH of the vacuole. Read on to learn more about prior methods for studying autophagy ...the vacuole via the secretory pathway, and then cleaved to generate an active form of the protein. To monitor...lysosome, since both Atg8p and LC3 are degraded or released once an autophagosome fuses with the vacuole or...

...requisite for a functioning lab, but it is for a scaleable lab. When you need to track the location, quality...post we’ll highlight some of the lessons we’ve learned over the years with the hope that our insights ...containers and contents Right from the get-go be as clear as you can be (there will always be confusion about..., tightly coupling them to their contents might leave you sad and confused when a tricky procedure failed...worm that requires a change of tube, there’s no clean way to separate the contents from the container ...etc.There are also going to be situations where you learn biology terms (plasmid, 5-prime, CRISPR, growth ...krisshamloo.com     Additional Resources on the Addgene Blog Learn about How We Use Barcodes in the Lab Get Tips on...

...the Addgene Blog Learn How Scientists Are Trying to Shape the Future of Research Learn How Notions of Scientific...studies with concrete examples of how gender analysis leads to discovery and innovation. One such case study... of their cells when doing experiments—which can lead to failed research. For example, an international...develop represent the whole human population. To learn a little more about where differences in effects...There is much work to be done! Researchers need to learn sophisticated methods of sex and gender analysis...Blogger Londa Schiebinger. Londa Schiebinger is a leading international expert on gender in science and technology...

...Ask openly for clear information on costs. Can you suggest some coaches? Yes I can. Please note that Addgene...to change jobs? Grow your professional presence? Learn a key new job skill? Finish your thesis? Knowing...that will help you. A good coach usually will be a clear communicator and make direct suggestions for improvement...She is also a very popular speaker and workshop leader. Lauren Celano of Propel Careers is not a professional...engagements and has many areas of specialty from leadership development to mentoring for women and under-...Guide Your PhD with An Individual Development Plan Learn a Little about The Academic Labor Environment from...

... 2019). Inhibiting DNA cleavage Example: AcrIIC1 binds to the HNH endonuclease domain of Cas9 and prevents...prevents target DNA cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can...loading, inhibiting DNA binding, or inhibiting DNA cleavage.   Five ways to anti-CRISPR proteins in your...timing experiments, the Doudna lab found that at least 50% of on-target Cas9 edits happens within the first...effects and cytotoxicity that result from excessive nuclease activity in tissues (Li et al., 2018). 4. Selection...Blog First time CRISPR user? Check out this post. Learn how to Choose the Best Cas9 Variant for Your Next...

...anymore. Say hello to PAM-free and PAM-flexible nucleases! In this blog, we will review Cas enzymes, which... uses a PAM with no sequence constraints, or at least has looser or different requirements. Luckily, there...Nonetheless, in an AT-rich region, these Cas9s may still leave you choosing between efficiency and location.   ...display higher off-target effects than SpCas9, a pleasant surprise since the PAM flexibility could in theory...activity of SpRY was validated in double stranded cleavage and base editing applications, with high editing... NG Cas9 NG - SpG Cas9  NG  - SpRY nuclease  C to T Base Editor SpRY A to G Base Editor SpRY...  Nishimasu, H., et al.,Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361,1259...

...implanted into a female animal for gestation. Nucleases that induce double-stranded DNA breaks, such as...cells, which are then injected into a blastocyst. Nucleases can also be used to engineer knockout mice. ZFN...non-homologous repair at the site of DNA damage. These nucleases are microinjected into a zygote, which is then...introduce point mutations. While using targeted nucleases to generate an engineered mouse model has advantages...targeting vector alone, some challenges still exist. Nucleases may cause off-target effects on regions of the... random process, zygotes edited using the same nuclease may not result in genetically identical mice. ...about Cre-lox Read our Genome Engineering Guide Learn more in our CRIPSR guide ...

...mEosFP exposure to UV light causes an irreversible cleavage near the chromophore, resulting in emission of...to blue light also inactivates the chromophore, leading to a non-fluorescent state. Dronpa can be reversibly...quickly inactivated and less photons are emitted leading to dimmer fluorescence. In fact, many of the early...are being emitted, making it easier to capture a clear image above background. The majority of PA-FPs come...disrupting cellular function and localization, or leading to protein aggregation. Another important parameter...light used to activate the first PA-FP does not photobleach the second, and vice versa. Finally, as every...contrast, PA-FPs offer a reduced potential for photobleaching and phototoxicity that standard FPs might induce...

...that delivering RNA-guided nucleases or TALENs together with AdV donor DNA leads to a vast majority of AdV-modified...chromosomal region. The CRISPR/Cas9-derived RNA-guided nuclease-induced gene disruption frequencies in the various... and demonstrated by Manuel Gonçalves and his colleagues at Leiden University Medical Center are now available...DNA for accurate genome editing with engineered nucleases. Nat Methods 11:1051–1057 . https://doi.org/10.1038...Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse...delivery of CRISPR/Cas9. Journal of Controlled Release 298:128–141 . https://doi.org/10.1016/j.jconrel...

...including mitosis, mitochondrial fission/fusion, nuclear import, and neuronal trafficking. Although FPs ...Overexpression of fluorescently tagged proteins can lead to improper protein localization, protein aggregation...power of this system lies in the ability of the endonuclease Cas9 to create a DNA double stranded break (...the tag, selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the...directed repair after a CRISPR/Cas9 induced break. Learn more about these constructs and the cell lines they...Cas systems used for endogenous protein tagging, please visit our website CRISPR/Cas Plasmids - Protein...PMC4889673. Additional Resources on the Addgene Blog Learn about Fluorescent Protein Timers Check out Photoactivatable...

...DNA could lead to transgene expression in recombinase-negative animals, producing misleading results.  ...the plasmid used to create the viral prep. In a clean viral prep, like AAV-44362 shown below, more than...from these known impurities, as was the case for clean sample AAV-44362. In addition to these expected ...number of hits to the same sequence (gene). In a clean sample, there are typically fewer than 10 hits to...receive the best AAV vectors for your research. Please use the comments section below to let us know if...Topic Page Subscribe to Viral Vectors Blog Posts Learn How We Use NGS for Plasmid Quality Control Resources...

...This DNA degradation portion can be bypassed by leaving out Cas3 — a tactic some CRISPR transposase systems...chop DNA. Cas3, however, has two jobs, one as a nuclease and one as a helicase. Following recruitment by...initial steps, Cas3 can perform its helicase and nuclease functions simultaneously, by continuously unraveling...unraveling dsDNA, pulling through the ssDNA, then cleaving the ssDNA strands. In this way, Cas3 eats away... into think it’s more powerful than it is. The nuclease domain of Cas3 is considered weak, especially ...However, by continually passing the ssDNA past the nuclease domain, Cas3 is able to repeatedly cut the DNA...thymine-rich regions on the non-target strand. Occasional cleaving of the target strand is not as well understood...

... PITCh plasmid to release the cassette, and an insertion-locus-specific gRNA cleaves genomic DNA. These...it’s become clear that the power of this editing platform lies in the diversity of nucleases and their .... For more mechanistic details on HDR and NHEJ, please see the linked blog posts. Compared to its counterparts...compare to CRISPR sticky-end insertion using the nuclease Cpf1. Since Cpf1 cuts in a staggered pattern, ...Experiment Additional Resources on Addgene.org Learn All about CRISPR on Our CRISPR Guide Pages Find ...

... Developed by David Liu and colleagues, FokI-dCas9 expresses Fok1 nuclease domain fused to catalytically... to this system by engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing...control for your experiments. For more information, please see Addgene’s information page for the Rinehart...neuronal signaling. For more information on DREADDs, please see the Roth lab's DREADD users blog. New ... expression plasmid that was used by Chen and colleagues for genome editing inXenopus tropicalis. (Guo...information on using the Platinum Gate TALEN Kit, please see the Yamamoto lab’s protocol for TALEN construction...mGRASP A Nature 2012 article by Jinny Kim and colleagues describes their efforts to map the location and...

...editors. They adjust the side groups of DNA bases and leave the core ring structure intact, allowing them to...guide RNA to base pair with the DNA target strand, leaving the non-target strand unpaired and accessible on...it the “edited strand.” Uracil excision repair leads to the first base transversion editor The story ...improved the CGBE: DNA polymerase D2 (POLD2), exonuclease 1 (EXO1), and RNA binding motif protein X-linked...included in the CGBE. They also developed a machine learning model capable of predicting editing outcomes of...that were already accessible. It's not entirely clear why this is, but it might point to a more dynamic... screens, target-library analysis, and machine learning. Nature Biotechnology, 39(11), 1414–1425. https...

...to see the dirty laundry exposed here, but it’s unclear how much they will actually care. The section of...technology. Though these case studies are “old,” they lead Zimmer to one of his most critical observations:...detection had over 1,000 authors. The chapter on deep learning addresses the dilemma of creating a technology...discussed The State of Science with the five Addgene colleagues who read it with me, the consistent response ...non-scientist alike was that the book left them wanting to learn more. There are individual paragraphs that may have...task to set oneself. As a scientist who enjoys learning a little bit about many different things (part...about a long time. For the scientists who like to learn everything about one specific topic, The State of...

...several washes, this tag can be cleaved by the bdNEDP1 protease, thus releasing purified nanobodies from the...common protein purification methods. Read on to learn more about nanobodies and how their structure and...primary antibodies raised in different species, or at least from different IgG subclasses, so that secondary... and the target antigen to about 2 nm, which can lead to higher resolution for super-resolution microscopy...rabbit nanobodies. If you’re interested, you can learn more about how nanobodies are identified in this...University Use Nanobodies to Control Protein Activity Learn more about protein tags Read about E. coli strains...

... clinical gene therapy applications. Read on to learn more about the current (and future) applications... co-transfection of these plasmids, 293T cells released transgene-containing lentiviral particles into...and they showed that expression persisted for at least thirty days. Although adeno-associated viral vectors...activation from the 5’ LTR) and Rev (facilitates nuclear export of transcripts). A different viral envelope...safer, this system may be more cumbersome to use and lead to lower viral titers due to higher plasmid number...Addgene Blog Your Lentiviral Plasmid FAQs Answered Learn How Lentiviral CRISPR Libraries Enable Genome-Scale... on Addgene.org Check out Our Lentivirus Guide Learn about Virus Biosafety Find Lentiviral Vectors for...

...journals. In addition, it is my pleasure to interact with my colleagues in meetings of scientific societies..."Biology is Method", i.e. new technologies often lead to big discoveries in biology. Therefore, my lab...translocation trap (ITT) system to analyze signal-induced nuclear translocation of proteins in 2004. In addition,...much enjoyed interactions with many excellent colleagues at NYU and visiting scientists, such as seminar..., I've been very lucky to work with excellent colleagues in my lab. On the other hand, I feel that researchers... conventional research. So, I hope my younger colleagues may be able to develop new technologies, hopefully...