...Without binding to the guide RNA, Cas9 cannot cut. 2. Bind to target DNA in the presence of a guide RNA...express a gRNA designed to direct Cas9 to their target sequence of choice (see tips for designing a gRNA) and... of the major CRISPR lingo and will be able to describe the functions of the various CRISPR/Cas9 components...responsible for processing foreign DNA as well as the RNA guides required for endonuclease function, when used for...Bind to a guide RNA The guide RNA, which will be described in more detail below, enables Cas9 to cut a specific...DNA damage is a DSB within the target DNA ~3-4 nucleotides upstream of the PAM sequence. Cas9 Species/Variants...with the appropriate PAM sequence) to modify the desired genomic locus.   Some historical notes CRISPR ...

....        Fig. 2: Reference image (left) provided to our graphic designer upon request that the...up walls of text. Since our guides are heavily used, our graphic designer made most of the Antibody Guide... quickly." Final design (right). Immediate and immense regret of the graphic designer not shown.   ...blog, website, social media, videos, newsletters, guides, and e-books – comes into play. Our goal is to ...communication between our content team and our graphic designer to make sure things are clear, accurate, and in...

...Nano-lanterns (eNL) described in Suzuki et al. improve the brightness of previous Nano-lanterns by 2-4 fold and... has precluded widespread adoption of the technology. In 2012, Saito et al. described the first Nano-...molecular and cell biologists over the last two decades, but their use can be limited by a few caveats....substrate furimazine. In addition to the previously described applications, the enhanced signal of eNL allows...autofluorescence and a high signal-to-noise ratio. Despite the many benefits of Nano-lantern technology, there...

...peer-mentoring groups at Addgene have gone virtual!   2. Practice public speaking For almost any science career...harder to build relationships when you are more desperate or needy so you can’t wait until you imminently...

...yourself greater chances for success.  Build a team of 2 or 3 Don’t go it alone.  You’ll find that no matter... own experiences founding and running miLab to describe some key lessons I learned while becoming a health...

... and whether I would be able to carry out a yeast-2-hybrid library screen; instead I thought they would...Coordinator’ came up at Abcam. Unlike other job descriptions for marketing positions posted on recruitment...

...MagFRET: The First Genetically Encoded Fluorescent Mg(2+) Sensor. PLoS One. 2013 Dec 2;8(12):e82009. doi: ...beginnings of the field. Those engineered sensors were designed to replace synthetic calcium dyes, which had been...

...automated. Unlike gel electrophoresis, you only need 1-2 ul of sample and the run time is just a few minutes...store your plasmid DNA Learn about the different grades of plasmid DNA preps Resources on Addgene.org ...

...created for the Brunello library is shown in Figure 2. You can use a small PCR product because the plasmids...next-generation sequencing (NGS). For gRNA libraries, you’ll design primers to create 200-300 bp sequencing products...

... remember that a resume is typically no more than 2 pages long. Write your resume for the job posting ...University of British Columbia, Student Services - includes videos on general resume writing, as well as additional...

...axon terminals to the soma (retrograde tracer) (Fig. 2). Viruses as neuronal scalpels Viruses can be injected...trillion connections (synapses.)  In this post we’ll describe the AAV retrograde serotype (AAVrg) and how it... this can cause challenges for the experimental design as these vectors can exhibit spurious expression...efficiency can have a major impact on experimental design and interpretation of results. Take extra care ...References 1. Tervo, D. Gowanlock R., et al. "A designer AAV variant permits efficient retrograde access...

...account when simulating a digest as depicted in Lane 2. Controlling methylation Finally, you can control... NEB's Resource describing Dam and Dcm Methylases Discovery of DpnI Review describing dam-/dcm- E. coli...mutagenesis. During this process, incorporation of a desired mutation into your plasmid of interest by PCR generates...only the non-mutated and methylated template is destroyed leaving behind a pool of mutated plasmids which...

...responsible for that differentiation.   Fig. 2: Single cell RNA-seq clustering of cells transduced...transcription factor library The human genome encodes approximately 1,800 TFs, with >3,500 unique isoforms... and screening mechanisms, although the unique barcodes for each TF help streamline identification. While...a disease in a model system. TF screens can be designed to identify custom TF programs to acutely initiate...

...perceived climate change risks. Nature Climate Change, 2(10), 732-735. doi:10.1038/nclimate1547 Kunda, Z. (...abstracts were driven by the quality of the study design or by their preconceived ideas about the controversial...transmission and how different fabrics and mask designs could work to slow the spread of illness. I later...Series: Masters of SciCommSummer Scicomm Series: Modes of CommunicationCareers in Science Communication...

...aspects of the work you presented may also be unclear. 2. Make the audience your primary focus When writing... craft sentences for abstracts and presentation slides, learn from audiences as they do science outreach...about whether the same principle applies to other slides in your deck.  If someone asks a question about...of a new science communication initiative that provides yet more scientists with opportunities to practice...

...types within a sample to expected labeling (Figure 2). The panel of antibodies used in this approach should...the antibody in the same tube.     Figure 2: Schematic showing the use of an orthogonal approach...see: Uhlen et al., 2016). In this post, we will describe the approaches that can be used to determine selectivity...intended application — here, flow cytometry — is the desired target, using one or more of the approaches outlined...interpreting your data therefore necessitates careful design of RNAi sequences, confirmation of RNA knockdown...the target at different levels, it is possible to design experiments where the performance of the antibody...We find that cell tracker dyes are helpful in designing such panels, so that pre-stained cell lines can...

...work with Grows extremely fast (doubling time of ~ 2 hours) Amenable to genetic manipulation The first...biomass production techniques in the following decades, Phillips made the decision to release the K. phaffii...of K. phaffii lacks certain terminal mannose glycopeptides (which most other yeast possess) that are problematic...

... 293HEK Inhibits termination step of translation 2-10 ug/mL G418/Geneticin neo HeLa, NIH3T3, CHO, 293HEK...mammalian plasmid components, it may be useful to describe the means of introducing genetic material (such...the plasmid in the same way. Selection methods, described below, are typically employed when creating stable...

...retransduce a model organism such as mice, e.g. first at 2 months, then at 4 months? Or do antibodies become ...1x1012 vg/mouse is more standard) and reflects a desire for very high MOI on top of very high coverage....researchers have used the flexibility of AAV genome design in remarkably creative ways. The continued development...

...Nutr, 129 (2009a): 477–484. PubMed PMID: 10064313. 2. Chapman-Smith, A., & Cronan, J. E. “The enzymatic...26224555. PubMed Central PMCID: PMC4884002. 10. Boer, E. de, Rodriguez, P., Bonte, E., Krijgsveld, J., Katsantoni...Northern blotting Cell surface labelling Attaching polypeptides to a substrate Flow cytometry/fluorescence-activated...your downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications...commonly used this method to attach proteins or polypeptides to a solid substrate (Holden and Cremer, 2003...