...heat-fixed smear with the crystal violet stain for 1 minute. Rinse the slide briefly with tap water. Flood...diderms, have a thinner peptidoglycan membrane and an outer lipid membrane. Common gram negative bacteria include...within their membrane, while the stain is washed from the gram negative bacteria’s thinner membrane. Procedure...destroys the outer lipid membrane, and the thin inner peptidoglycan membrane is unable to retain the stain...expanded on Gram’s discovery, adding a second stain, safranin, as a counterstain to help visualize the bleached...or monoderms, have a single thick peptidoglycan membrane. Common gram positive bacteria include staphylococci...and immediately rinse. Flood the smear with the safranin counterstain for 30 - 60 seconds. Rinse the slide...

...Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure 1). Once introduced, fluorescent...fluorescent protein (XFP) genes in a transgene. When multiple copies of the transgene cassette are introduced, stochasticity...fluorescent proteins is only possible at low copy number ranges, resulting in low fluorescent protein level. The...gene delivery is more common than conventional transgenic strategies. In such vector-based gene delivery...in the original method, we used the Tetracycline trans-activator system to boost the expression levels,...expression level of XFPs, most likely by suppressing transcription. It is critical to express a minimal amount ...Bio-Protocol 8 (2018): e3046. Livet, Jean, et al. "Transgenic strategies for combinatorial expression of fluorescent...

... Figure 1: Identification and enrichment use of XMAS-TREE. a) Cells are transfected with three different...ready-to-use viral preps   Improved cell-free transcription/translation system to examine biosynthetic pathways... Gabrielle Clouse Recently, cell-free transcription/translation systems (also referred to as TX-TL systems...modifications to edit DNA without requiring a double-stranded break. The Xiao Wang Lab has recently deposited.... These plasmids use “Cas9-mediated adenosine transient reporter for editing enrichment”, or XMAS-TREE...identification of essential MYC binding sites in a wide range of human cells.  A new pooled library from Junjie...

...construction of redCALWY-1, a red-shifted FRET sensor for zinc using variants of mOrange2 and mCherry as donor...field is a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors. Perhaps most ...developed and published in 2010 for visualizing the transcription factor cAMP-responsive element-binding protein...really important and when? That is not known.” Transition Metals Maarten Merkx of Technische Universiteit...particularly for the intracellular imaging of transition metal ions, such as zinc, copper, and iron. “...the sensors involves plenty of trial and error. Transition metals also present challenges in that they are... way to make a FRET sensor with a large dynamic range,” he says. Merkx recently applied this “trick” to...

...preferentially). You can also rank the plasmids by abundance, scaled to 1, to create a “cumulative wealth... seems straightforward: E. coli bacteria are transformed with the library DNA, they grow and replicate... Each Addgene library has its own depositor transformation and amplification protocol, but the overall...Maintain representation of the library during transformation First, use enough DNA and competent bacteria...and we use electroporation to ensure a good transformation efficiency. For a typical library, Addgene ...growing the cells on solid plates, where each transformant forms its own colony, uninfluenced by the growth...concern, but Addgene still recommends plating the transformation mixture on solid media. The colonies are then...

.... You can email her at [email protected].   References 1. Huang, Ally, et al. "BioBits™ Explorer: A modular...that green fluorescent protein through transcription and translation. Unfortunately, having live cells in... like gel electrophoresis to separate DNA or transforming cells with green fluorescent protein, these ... into a host cell (through a process called transformation), essentially giving the cell all the instructions...enzymes that can convert certain chemicals into fragrances that they can smell, or enzymes that can create...perhaps consider using some elements of BioBits to transition your undergraduate students from the classroom...

...occur, but good performers move around easily  Table 1: Taking it a Step Further - Big Company v. Small Company...encompasses millions of choices including pharma, tech transfer, management consulting, science communication,...into industry.” In his article On Leaving Academe, Terran Lane is asked by his colleagues and friends why...tell you to pick up the pace Constant worry about grants and renewals What is different about non-academia...outsource a task someone else who can do better, no grant writing Job changes can happen regularly (every ...where you want to go will help you focus on the transferable skills that you will need to get there. Additional...

... contain the full length plasmid.   Figure 1: The full lentiviral plasmid is expected to be >12...recombination, one resulting plasmid carries the transgene of interest (aka the viral genome) and the other...resistance gene and origin will replicate, and the transgene will be lost. Over time, bacteria containing the... grow! Our 5 Tips for Troubleshooting Viral Transductions will help with the next steps.   References...recombination of plasmid DNA without affecting mRNA translation efficiency or half-life. RNA 25:507–518. https...common lab E. coli strains Find tips for viral transduction  ...

...motion videos is very time consuming; producing a 1-2 minute video takes an entire week of our free time...have the same issues as one doing experiments in France. We can learn with each other through globalized...

...class.   Four ways to improve your next manuscript 1. Be consistent with your figures The figures of a ...worthless. But simply writing down your data doesn’t guarantee a publication. There is an art to composing manuscripts...

...then scroll on for some wintery delights!    Table 1: New ready-to-use viral vectors in the Addgene repository...Request service, which offers over 3,000 eligible transfer plasmids and five different serotypes. Not sure...

...heterodimerizes with CIBN upon blue light exposure (see Fig 1*). When the group targeted the enhancer region of ...ubiquitin ligase, the Kanemaki lab was able to translate AID to yeast, as well as mammalian and human cell...PubMed PMID: 31026591. Expanding the targeting range of CRISPRCas9 by engineering SpCas9 Article contributed...are also thought to play a key role in the transcriptional regulation of many genes. A number of groups...thus far for these latter experiments, such as transcription factors tethered to dCas9, require long time...

...wide range of input DNA concentrations. This process is explained more fully in the animated Fig. 1 below...plasmids on a plate) will typically have a wide range of input quantities. This variation is problematic...produced from large numbers of samples with a wide range of input concentrations (via proprietary chemistry...differences in input concentration across a 100-fold range (1ng – 100ng) are normalized to two-fold variation...

...produce as many as 700 copies per cell. Figure 1: A plasmid map showing the standard features of a ...plasmids from the same group should not be co-transformed, so if you need two plasmids for an experiment... same incompatibility group should not be co-transformed. +Actual copy number varies. See below for ...stocks, agar stabs, or liquid cultures. Freshly transformed cells also give a higher plasmid yield that colonies...

... and can likely be used in a wide range of bacteria. References 1. Maddalo, Danilo, et al. “In vivo...post about multiplexing with Cpf1.   Figure 1: Multiplexing allows researchers to express multiple...humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing...Multiplexing from a single transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being...being transcribed from different promoters, the gRNAs are transcribed together and are flanked by specific...simultaneously. Vectors for both transient expression and Agrobacterium-mediated transformation are available. For ...system for processing a continuous multi-gRNA transcript, each gRNA must be expressed from its own promoter...

...Article contributed by Susanna Bachle Figure 1: Initial steps in the CRISPR-pooled screening using...Sabrina Büttner’s lab designed a calcineurin transcriptional reporter consisting of a quickly-degraded destabilized...contributed by Jennifer Tsang Figure 4: Double stranded DNA bound by tTALE-FP with the fluorescent protein...

...net negative charge with 4 negatively charged and 1 positively charged amino acids. Myc tags are frequently...some proteins. Protein folding issues, steric hindrances, and destabilization can be brought about by ...DOI: 10.1038/521274a Arribere, J. A., et al., Translation readthrough mitigation. Nature. (2016) 534, 719...

...extension. Now, they split EB1 into two halves (Fig. 1): one containing the microtubule binding domain and...manipulations on cells, and has potential use for a wide range of applications in cellular biology. Van Haren ...fluorescent proteins (FP) with improved tissue penetrance and live cell imaging capabilities. Upon developing...

...defunct (but fun) plug-in called Maps.  Figure 1: See a starfish? This is actually a map of Joanne ...enough, but I’ll never forget the strawberries. Grand gestures are memorable! Attend events for one organization... want to do: Don’t connect via LinkedIn with strangers. It’s a waste of time. People that don’t know ...

...impartial. The best science happens when scientists have 1) the courage to make mistakes and 2) the integrity...of what I saw are that we, as a society, need to: 1) use more citations for out data so our sources are...I’ve worked tirelessly to get the publications, grant funding, and teaching experience that it takes to...