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...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...

...the reporter mRNA, which contains the tethering sites for the fluorescent proteins and is driven by an... copies of the PP7 and MS2 coat protein binding sites to tether many copies of the FP-coat protein fusions...NLS-PCP-GFP is removed by the first ribosome that translates the mRNA. To permit tracking over time, Yan et...Each time the reporter is translated, the SunTag sites on the nascent polypeptide recruit scFV-GFP, generating...translation in various cell types and physiological states. References 1. Ingolia, Nicholas T., et al. “Genome-wide...SunTag Catch up on Your GFP Background Choose the Brightest Fluorescent Protein Resources on Addgene.org ...

...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. ...

...majority of research institutes.  Pro tip! In most academic and nonprofit institutes, the Tech Transfer ... signatures from two different institutes. Our online portal automates a lot of the process, from notifications...  An MTA is simply a contract or agreement that states the terms of use for a tangible research material...people think our MTAs are different than other institutes. But they’re not! The vast majority of our plasmids...

...yield, and when transiently expressed in cells, generates much less signal than comparable fluorescent proteins... very similar to the widely used FRET, but it eliminates the need for an excitation light source. Instead...multiple subcellular compartments over several minutes using Nano-lantern fusion proteins. To further ...continuously imaged cells for about 4 hours! The latest enhanced Nano-lanterns (eNL) described in Suzuki...fluorescent imaging! These eNL constructs use the brightest characterized luciferase, NanoLuc, and its substrate.... Filtering the emission wavelength partially separates the signals of various Nano-lanterns, but it does...

...Why do scientists use yeast vectors? Yeast are eukaryotes and thus contain complex internal cell structures...technical advantages that come with working with prokaryotes. This includes but is not limited to: rapid growth... selection when working with yeast, due to high rates of spontaneously occurring resistant mutants and...complementation is weak. yes   LYS2 L-lysine yes - Grow on plates containing alpha-aminoadipate in the absence of...physiological levels due to high copy numbers. This creates a potential metabolic burden on the yeast cells...markers. The MX series of antibiotic resistance cassettes is most commonly employed, with the KanMX being...

...microscopy, a metal disk containing a number of holes rotates through the excitation light path. Each hole corresponds...happen.Using red light has a second advantage: red light penetrates deeper into biological tissue. To prove this,... create a thin sheet of excitation light that propagates perpendicular to an imaging objective that collects...into the sample via an evanescent wave that only excites fluorophores within a few hundred nanometers of...Fluorescence (TIRF) microscope can be used. TIRF only excites fluorophores within a few hundred nanometers of... pixel by pixel are unable to provide the frame-rates necessary for imaging tissues of this size. For ...Fluorescent Proteins for Multi-Color Imaging Choose the Brightest Fluorescent Protein for Your Experiments Resources...

...and MEK1 is active or “on.” Dronpa’s color thus indicates MEK1’s current state (Green when off, dark when...photoswitchable Dronpa domains were inserted at sites homologous to the locations they were attached to... a few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the ability...indicator of MEK1 activity. When MEK1 is active, it phosphorylates the ERK KTR and red fluorescence localizes ... fluorescence can be found in the nucleus. For testing kinase inhibitors, psMEK1 and ERK KTR-mRuby2 expressing...blot. Phosphorylation of MEK1/2 peaks at just 5 minutes post-Raf1 inactivation, and then decreases. Treatment...

...autoinflammatory loop: keratinocytes both respond to and release IL-36 cytokines. Keratinocytes are the main source...cytokine family resulting in the activation of keratinocytes, which release inflammatory mediators that trigger...known how IL-36 cytokines actually get out of keratinocytes and induce their effector functions. It’s an...important to study what is happening within the keratinocytes. “We hope to use CRISPR libraries from Addgene...or proteins are involved in IL-36 release in keratinocytes,” says Mellett. This would help the lab identify...

...’s not, I can always take more time to check my notes!  Know your purpose The purpose of our assignment...assignment was clear: present a paper to our classmates so they could understand it. When my mind went blank,...gene of interest incorrectly on slides 8-16. Your notes disappear. The wrong version of your slide deck ...simple as telling yourself, “I can stop and check my notes if I need to,” or “It is okay if it doesn’t go perfectly... field, with very little text on the slides and notes instead of written speeches. When you’re doing all...speak at conferences, or even just provide quick updates during a company meeting increases visibility of...

...a substrate promoting dimerization, Tang et al. tested pairs of their GFP-binding proteins (GBPs) to find...Transcriptional Devices Dependent On GFP). In testing the specificity of their system, Tang et al. found...was absent without electroporated GFP. They then tested T-DDOGs with two mouse GFP reporter lines; again... activation frequency of 56-93%. In the converse test, 98% of TdTomato expressing cells were GFP+, indicating...recombinase, which induces recombination at LoxP sites. Placing a lox-STOP-lox cassette upstream of a gene...T-DDOGs, Cre-DOG is both robust and specific. When tested in retinal electroporation studies, DsRed reporter...

...technical writing and communication skills, Elucidates the big picture and future directions of the ...milestones, possible setbacks, and potential alternative routes, Provides opportunities to convince others why...It typically includes overview information, key dates, funding description, research plan, award information...institution. Moreover, avoidance of ‘to be’ verbs creates strong, well-directed meaning and delivers strength...section. Research challenge The Research Challenge motivates the request for application and offers an opportunity...reaching, remember that you are writing a proposal to test hypotheses and develop novel technologies! If data...

...So, when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9...disease: cancer (Feinberg & Vogelstein 1983). The floodgates, as they say, had opened.  In 2021,  with over...these studies report correlations without clearly testing whether a certain methylation state causes a condition... we need tools to specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase...independent of dCas9 targeting, including modifying other sites in the genome and directly interacting with transcription...

...which the story demonstrates I possess.   Bullet points on resumes are like cliff notes, so I use these... interviews. I’d also use this notebook to take notes after each interview.   Tailor your 1-hour presentation...rushed and you’ll have to answer questions between bites of food.  Act and look professional  First impressions...and others, if you have their emails. You can use notes you took during your interviews to help craft these...technical difficulties. To try and circumvent this, test out the video conferencing software you’ll be using...

...can mutate the transcription start site, splice sites, exons, and/or promoters to knock out lncRNA function...Bester 2021; Zibitt et al. 2021). Mutating splice sites is particularly effective as it can result in the...Knock-in: Introducing tags or stop signals Once Cas9 generates double-strand breaks, a new DNA sequence can be... be knocked-in, or introduced, into the break sites. Knock-in experiments are important for visualizing...start site (TSS) deletion. (B) Mutations of splice sites. (C) Removal of an exon or a large genomic fragment...RNAs in human cells by Cas9 targeting of splice sites. Nat Biotechnol. Published online November 5, 2018...

...an additional 5–10 minutes of incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh wash...solution and agitate for 30–45 minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer...respectively. Chromogenic detection produces colored precipitates that permanently stain the membrane, making ...membrane in stripping solution and agitate for 5–30 minutes at room temperature. The incubation time will depend...

...proteins that cause rare diseases. They now plan to test a large number of other disease-linked Arg-mutant...Engineering the human disease alleles into cells for testing in the lab will be greatly facilitated by CRISPR...Baldwin, Enoch, et al. "Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions...function. They will then mutagenize the NFIX gene and test whether variants of unknown significance (VUSs) ...) that are frequently found in genetic testing for syndromes that include developmental delay, result ...dominant negative NFIX phenotypes. Developing a rapid test for the functional activity of VUSs will enable ...

...that blood flows through the heart and lungs and oxygenates the body. However, in neuroscience, this architecture... between neurons are what define how the brain operates, and thus, are a major part of the answer to many...infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross synapses...necessary and sufficient for retrograde transmission (Etessami et al., 2000, Wickersham et al., 2007). Thus, ...answers to the complex questions of how the brain operates. References 1. Callaway EM. Transneuronal Circuit...Mar 19;110(12):E1152-61 Pubmed PMID 23487772. 3. Etessami R, Conzelmann KK, Fadai-Ghotbi B, Natelson B, ...

...cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to Kim et al. ...in AT-rich genomes. Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1... strength may lie in its low off-target editing rates, determined using sophisticated genome-wide analysis...many of its computationally predicted off-target sites, Cpf1 does not mediate detectable off-target cleavage...mechanism. To prevent new editing post-HDR, repair templates should remove the PAM sequence. Many research ...

...recommend no more than 40 minutes for the panel discussion leaving 20 minutes for audience questions (30...Follow Addgene on LinkedIn for repository news and updates. Panel discussions are becoming a ubiquitous format...members all say the same thing and one panel member dominates the discussion? Or maybe the one that goes way...interviews, the moderator should be taking good notes and discovering interesting stories, riffs or opinions...be good at talking, so they will easily take 5 minutes to introduce themselves and, voila, 1/2 of your...