...Chapter 1 asks “What is science?” That’s a very big question. Zimmer, a Professor of Chemistry at Connecticut...chapters in this section describe the challenges of trusting science in a “fake news and post-truth era,” the...would be complete without the inclusion of some interesting case studies, and Zimmer provides several. In...throughout this chapter. Now, these observations and suggestions are not so much inaccurate as already somewhat...period of time. Though climate change certainly still is and will continue to be a “critical consequence...current period of rapid flux, the chapter might still feel slightly dated by next week.   A stepping stone...

...correctly, there’s no point in crystallizing it or testing its function. Small ubiquitin-related modifier .... Arguably the simplest affinity tag is the polyhistidine (His) tag. Small and unlikely to affect function...the tag size quickly – modifying the length a polyhistidine tract does not greatly alter the size of the...biotinylation signaling peptide (Bio), flanked by hexahistidine motifs (6xHis) MBP Large Protein  40 Solubility...combinations, though frequently those combinations still include at least one element from the original TAP...purpose, and you don’t necessarily want them to stick around after that purpose has been served. At this...

... proactively address questions about your application. These could be logistical, like briefly explaining...and it does take a bit of time to craft one. We still think they're useful, so we've updated our cover...and used a ddPCR assay to answer an experimental question. Now, the hiring manager knows that you can design...most important skills for the position]. I am enthusiastic about [something that you love and is important...any unusual circumstances or proactively answer questions the hiring manager may have. The closing paragraph...

..., a pooled library has hundreds to millions of distinct plasmids, all of which need to be represented ...The size and composition of your library can drastically impact what conditions it needs to properly amplify...pre-made libraries aren’t quite plug-and-play: you’ll still need to titer and optimize before you can transduce...your research. And, of course, if you have any questions, don’t hesitate to email us at [email protected], this is a useful place to ask experimental questions.  Practical Considerations for Using Pooled Lentiviral...lentiviral CRISPR libraries. Protocols from the Broad Institute- A variety of protocols and guides regarding pooled...

...by enhanced green fluorescent protein (EGFP) reconstitution. (a) Scheme of validation for DSB mediated ...mediated EGFP expression cassette reconstitution. When the target sequence was digested by sgRNA guided Cas9 ...SSA, single strand annealing) took place and reconstituted the EGFP expression cassette. (b) pCAG-EGxxFP...details on this plasmid, see Mashiko et al., 2013). Testing the efficacy of your gRNAs is then a simple matter...of gene modification in vivo. Find Plasmids for Testing Your gRNAs in vitro at Addgene CRISPR/Cas9 gene...require you to house mice that may have no mutation, costing money and space. As a rule, screen all of your ...

...promote cell growth. Overgrown cultures stimulate quiescence, the resting phase of the cell cycle; quiescent...lentiviral infection, happy virus is the second. When harvesting lentivirus, some labs prefer to collect and pool...the positively charged polycation, polybrene. Interestingly, one study found that alternative polycations...routinely produce lentivirus may want to consider testing a variety of cation and serum sources to ensure... the best conditions for specific applications, stick with them! Changes in such variables as cell density...

... neuronal populations in the brain. The Allen Institute and the Janelia Research Campus in the USA have...classic probes. To overcome the limitations of existing FP and epitope tags, researchers in Loren Looger...set-up. In contrast, smFP tags can be recognized by distinct, fluorophore-labeled antibodies with very specific...smFP_FLAG than GFP.   3. Enhanced ability to distinguish weakly expressed and/or similar proteins Through...proteins than standard tags and can be used to distinguish highly similar proteins. For example, N-cadherin...terms of sequence and thus very difficult to distinguish using standard antibodies. By fusing N-cadherin...

...we are investigating how the production of cancer therapeutic drugs, vinblastine and vincristine, are regulated...expression method enables us to quickly screen and investigate transcription factor candidates and the regulation...transient methods are usually the first choice for investigating gene function, as these experiments can be done...genes and speeding up research. Certainly, there is still more to transient leaf transformations, but we hope...

... spatiotemporal control over its expression. Constitutive or inducible Cre recombinases can be introduced...coding regions. Typically, each gene isoform has a distinct transcriptional start site. Because CRISPRa can...long-term memory formation. The Bdnf gene possesses 9 distinct transcriptional start sites, all of which yield...singled out two Bdnf isoforms (I and IV) for investigation. Co-transduction of primary rat neurons with...notable exception) necessitates such molecular gymnastics for sensitive applications such as CRISPR screening...CRISPRa? Although cDNA overexpression approaches may still be preferable for individual gene activation in ...

...in what they can name a gene and many of the interesting ones come from the fly community. Not surprising...the Shivdasani Lab from the Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic...got its name, we need to go back to 1980, when Christine Nusslein-Volhard and Eric Wiechaus first identified...cytokinesis. Why name this protein after a vegetable substitute for spaghetti? I actually couldn’t find the specific...failure of primary spermatocytes to enter meiosis. Testis of cookie monster mutant flies are filled with ...1189. PubMed PMID:1905980. Nüsslein-Volhard, Christiane, and Eric Wieschaus. "Mutations affecting segment...

...echoed in the other testimonies we gathered. The program definitely caused a positive stir and was recognized...invaluable to me as a bench scientist working at an institution where training opportunities weren’t always available...were connected to that Tweet Up in 2013. These existing connections brought in folks I didn’t yet know....  They liked us. They really liked us.  The testimonials from participants in Binning Singletons at ASM...Heather Hendrickson, Jonathan Jacobs, Mo Kaze, Christina Kellogg, Heather Martinez, Pat Schloss, Tara Smith...

...BFP mutant (BFPH66) contains a histidine at amino acid 66. This histidine is encoded by a ‘CAC’ codon that...consists of a mutated mCherry and a downstream constitutively active eGFP. To create the inactive mCherry...T2A site and expressed in cells. mCherry is constitutively expressed to identify successfully transfected...C-to-T base editing event. This edit changes the histidine to a tyrosine generating a GFP variant (GFPY66...markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing reporter toolbox. ...

...creative, usually saying that they’re not artists, or artistic, and therefore not creative. However, if we think...2019) If you’ve taken the time to do this and are still struggling with idea generation, try this thought...to the same problem. If your lab/company/team is still working towards a culture of encouraging dissent...This is why perfectionists tend to also be procrastinators.  If you fall into this category, get out of...easier to work with than nothing at all - they’re still ideas, after all, and that is the goal of this exercise... paint like Frida Kahlo or you’re stuck drawing stick figures, remember your training as a scientist has...

...Leong Chew is a Junior Principal Investigator at the Genome Institute of Singapore. His lab focuses on...with Wei Leong Chew, a researcher at the Genome Institute of Singapore who recently started his own lab....work to an independent position at the Genome Institute of Singapore. Chapter 3: Biology research and ... Singapore. Chapter 4: Life as a new primary investigator. Coming up with new project ideas, applying ...

...multiple streams for composting and recycling in the event the object in question cannot be washed or ...reuse, reducing the need for the production of new plastic.  Recycling and reuse is not limited to the office... however. We participate in many styrofoam and plastic recycling programs in the lab as well. For a more...Practices to the Lab.”  Whether participating in an existing process or proposing new programs, we're always...

...advantage for researchers looking to generate long-lasting changes in cells. However, for some applications... genome. Without a working integrase, IDLVs can still deliver their genetic payload to the cell but cannot... IDLVs were first discovered by researchers investigating HIV integrase mutants in the early 1990s (Leavitt...in several patients developing T-cell acute lymphoblastic leukemia due to insertional mutagenesis (Howe...avoid the risk of insertional mutagenesis while still allowing for stable expression of the transgene....of SCID-X1 patients. The Journal of Clinical Investigation, 118(9), 3143–3150. https://doi.org/10.1172/...

...cells on a flow plot, you might have to do some testing or ask experienced colleagues.   Figure...flow analysis I can spot my lymphocyte population distinct from cellular debris and other larger cells. Note...following gates, cells that are not of interest can still be gated out later. Based on personal preference... many cases a stain will not result in clearly distinct positive and negative populations. Oftentimes,...read an FSC/SSC plot and how to use H vs. A to distinguish single cells from duplicates. Lastly, you learned...Harvard Medical School. His projects include investigating negative regulators of T cells and cytokine ...

... the Institute for Protein Innovation (IPI), a Boston-based, nonprofit protein research institute, partnered...researchers can measure this conformational change to investigate the role of specific integrins in cellular processes...subunit. Using these affinity reagents, the investigators inhibited other RGD-binding integrins, individually...for cell adhesion. The results surprised the investigators, challenging the long-held belief that αVβ3 ...IPI. Caitlin Faulds is a science writer at the Institute for Protein Innovation. She writes about all things...

...mutations, we still weren’t entirely happy with our editing outcomes and in many cases still didn’t get ...must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway. Accomplishing this... be screening a total of a few hundred clones - Still better than 10.000, right? (Kwart, Paquet, et al...reads that we studied by deep sequencing, but interestingly (and to our frustration) in many cases we only...currently a Professor for Neurobiology at the Institute for Stroke and Dementia Research, Ludwig-Maximilians-University...

...cytosine-to-thymine substitutions in target sequences (Banno et al., 2018). These substitutions were limited ...enable C-to-T and A-to-G base substitutions respectively. Substitutions seem most efficient when using...interest, inducing a lethal DSB.     This system is distinct from those used in eukaryotes in that CRISPR isn...targeted and preferentially depleted from gut. While still far from the clinic, CRISPR-Cas3 systems have been... Sharp JD, Xu R, Prisic S, Husson RN (2016) Investigating essential gene function inMycobacterium tuberculosisusing...