...CRISPR gene activation system, but settled on a different optimal fusion protein combination. The optimized...AcrIIA4, this unfolding conformational shift can interfere with activity. The resulting system was coined...Alexander Deiters’ lab, on the other hand, took a different approach. These researchers used a genetically...expression - demonstrating light-induced silencing of transferrin receptor CD71 in HeLa cells.   Comparison of...applications you use these for in the future! References Bubeck F, Hoffmann MD, Harteveld Z, Aschenbrenner...photoactivatable transcription systems to induce neuronal differentiation. Nat Methods 14:963–966 . https://doi.org/10.1038...

...This intrinsic bacterial recombination pathway, referred to as recA-independent recombination, joins together...single PCR, for example for subcloning genes from different templates. All PCRs are run as an 18 cycle, 25...pre-made aliquots of PCR master-mix, containing buffer, polymerase and dNTPs, can be stored frozen: just... on Twitter @jakefwatson and @JGarciaNafria.  References 1. Jones, Douglas H., and Bruce H. Howard. "A...for plasmid cloning Find our molecular biology reference here Find molecular biology protocols here   ...

...the same figure by figure organization with a different person responsible for each figure. Others still...questions they have.  These variations are suited for different applications and should be carefully chosen for... and audio recordings of the meeting using teleconferencing software. Any file sharing service should ...or instant messaging but we as scientists often prefer data. At Addgene we rely on polls and surveys. ...acquisition of the essential data and allow everyone to offer thoughts you may not have even considered. These...

...written by Addgenie Brian O'Neill.  References and resources References Bauzon, M., Castro, D., Karr, M.,...vectorization strategies include creating an AdV transfer plasmid with the E1 and E3 (Early 1 and Early ...Although the late genes are usually present in the transfer plasmid for the majority of adenoviral vector ...B. (2014). Adenoviruses: General Features. In Reference Module in Biomedical Sciences. Elsevier. https...

... in and out of the classroom.   References and Resources References Bangera, G., Brownell, S. 2014. Course-based...the University of San Diego), decided to take a different approach. His own most impactful learning experiences...all shaped his teaching style, with the former offering excellent training in improvisation and the latter... available technical and pedagogical resources, offers mentoring for instructors, and provides opportunities...

...twitter @Vopinator.     References: Halpin-Healy TS, Klompe SE, Sternberg SH, Fernández IS (2020) Structural...Insertion of the mini-transposon can occur in two different orientations, but is strongly biased towards T-RL...combined in single-plasmid INTEGRATE (pSPIN), which offer simple transformations for smaller cargos, and the...integration efficiencies achieved with VchINT, at two different incubation temperatures. Data adapted from Vo ...

...denotes intersection and the – operator denotes set difference. That’s the intuition for how genome math helps...enhancers from step 2. See which ESCs die or differentiate Step 3 reveals which NANOG-related genes are...distinct enhancer disrupted. As our ESCs die and differentiate over time, we periodically use next-generation...Therefore if a gRNA drops or disappears over time, we infer that the enhancer it targets is a “pillar of function...by starting with all of the DHS sites from 125 different human cell types from the ENCODE Project. DHS ...

...viral infection), producing firefly luciferase. Firefly luciferase can readily be measured to quantify...single (non-viral) reporter gene (e.g. firefly luciferase or eGFP) flanked by the EBOV-specific gene start...to transcribe the reporter gene (e.g. firefly luciferase). The polymerase recognizes the gene start signal...transcription of negative strand RNA viruses.   References 1. Buchholz, Ursula J., Stefan Finke, and Karl-Klaus...

...moving around to explore the environment has a different motivation (and brain areas associated) compared...addiction. Anxiety tests Rodents have a natural preference to stay in dark areas (protected) as opposed ...to brightly illuminated ones (risky). They also prefer to explore novel environments by initially moving...anxiety. On the other hand, the control group preferred staying in the dark compartment (Howerton at al...these in a  future blog post, so stay tuned!  References Alpár, Alán, et al. "Hypothalamic CNTF volume...

...human pluripotent stem cells By Jennifer Tsang CRISPR gene interference (CRISPRi) and gene activation (...For labeling the plasma membrane, they tested different target sequences for improved selectivity and ...into the supernatant. Four plasmids containing different nanobodies (NB) were generated in pMAK:  pMAK461....1038/s41598-020-57500-1 The CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR...

...limitations associated with SpCas9. Cas13a (previously referred to as C2c2), has several unique properties that...for target recognition and cleavage). C2c2 (now referred to as Cas13a) is unique in terms of structure ...remainder of this blog post. Perhaps the biggest difference between Cas13a and Cas9 is that Cas13a binds ...SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika virus, genotype human DNA, identify...Gearing contributed to the update of this post. References 1. Abudayyeh, Omar O., et al. "C2c2 is a single-component...

... to determine the pros and cons of using many different types of gRNA design software. When you are happy...inserted or deleted. Each lab will use a slightly different method to genotype their pups. Taking a toe, a...weeks old and use the ear tag method (creating different patterns of ear holes and using that material ...latest and greatest in scientific content.      References 1. Cho, Seung Woo, et al. "Analysis of off-target...plasmid into zygotes." Development, growth & differentiation 56.1 (2014): 122-129. PubMed PMID: 24372541...

...C-terminus of the fluorescent protein. I prefer to describe the different parts of the fusion from N- to C-terminus...tagged protein because of its size. This type of interference is known as steric hindrance. For instance, ...advisable to generate multiple constructs with different insertions, since it is difficult to predict which...can follow him on twitter: @joachimgoedhart.   References Adjobo-Hermans, Merel JW, et al. "Real-time visualization...

...labs. While many labs approach the situation differently, some adopting simple note-taking tools, others...functionalities with experiment records. Many different types of ELNs are available on the market today... comparison to paper-based record keeping, ELNs offer functionalities that are hard to imagine on paper...desired functionalities i.e. main priorities of different ELN features, ranging from whether respondents... allows you to assign specific permissions to different lab members to avoid any issues later on. Do you...

...delivering scFvs that inhibit HIV.   References and Resources References: Ahmad ZA, Yeap SK, Ali AM, Ho WY...for high affinity scFvs. Bound phage are then proliferated by infecting bacteria for additional rounds ... high affinity binding. Bound phage are then proliferated by infecting bacteria for additional rounds ...science.3140379  Bradbury ARM, Sidhu S, Dübel S, McCafferty J (2011) Beyond natural antibodies: the power...

...interviews, or get several interviews but no job offers, is a failure to clearly articulate one's talents...to convince me that their search committee is different and needs to be told all of the nuances of the...technologies, i.e. video interviews. For point of reference, I submitted my applications all by snail mail...hoppy pint of IPA or a few drams of single malt (preferably from Islay or Skye). Good luck with your search...University, did his postdoctoral fellowship with Jennifer Lippincott-Schwartz at the National Institutes...

...of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as Cas9) and a single stranded guide RNA (gRNA...agrobacterium-mediated delivery. Summary While different in many of the specifics - promoters used, precise...mediated genome engineering in plants isn’t all that different from how it’s used in other systems. Luckily, ... don’t be afraid to try your hand at CRISPR. References Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov...of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol...

... Even low-frequency editing could make a huge difference in DMD, and it’s estimated that exon skipping...respectively. Although each study used slightly different methods, they each observed the same positive ...disease type C. Although the three DMD studies referenced here represent a great step forward for CRISPR...many distinct CRISPR therapies to match these different mutations. For each therapy, effectiveness and...treatments may also be closer than we might expect.   References 1. Kaiser J. CRISPR helps heal mice with muscular...

...bottom of this post for a basic synopsis of the differences amongst the Brainbow systems. Pre-Brainbow techniques...to the promoter will be expressed. This system differs from Brainbow-1.0 in that transient Cre recombination...needed to mark individual neurons, the system also suffered from a number of limitations. First, the imaging...controlled by FLP/FRT recombinase. When placed under different promoters, Brainbow and Flpbow can be used to ...2.1 plasmids Brainbow 3.0, 3.1, 3.2 plasmids References: Transgenic strategies for combinatorial expression...

...PiggyBac transposons, the Woltjen Lab compared different variants of the polycistronic cassettes and discovered...a donor in FRET (fluorescence resonance energy transfer) and FLIM (Fluorescence Lifetime Imaging Microscopy...is a new member of this class that works via a different mechanism - the PhoCl protein is irreversibly ... are other RFP variants available that may be preferred for their intended uses, they provide a well-defined...of essential genes in vivo. Utilizing CRISPR interference (CRISPRi) technology, the arrayed knockdown ...defined domains can be swapped onto other proteins to confer functionality. In Narayan et al., they used this...