...that no component of the overall project gets left undone or is duplicated. With each component of the ...the project completed by an individual who is a local expert in that task (or has the bandwidth to become...result is high quality and the entire process is completed more efficiently. Listen to our podcast interview...desired outcomes. Concern yourself with what is accomplished, rather than detailing how the work should be...in more engaged, successful employees who can accomplish great things.   To read more on this topic and...

...all bacteria are naturally competent to take up DNA, they can be made competent through chemical manipulation...Addgene’s blog to learn about making your own competent cells and our protocols page to learn about bacterial... The phagemids are packaged into replication-incompetent phage particles with assistance from a ‘helper... A., and Karen Guillemin. "Multiple phases of competence occur during the Helicobacter pylori growth cycle...Meibom, Karin L., et al. "Chitin induces natural competence in Vibrio cholerae." Science 310.5755 (2005):...

...fluorescent proteins, how to do a head-to-head comparison of fluorescent proteins and how to make a well-informed...your selection on a small-scale head-to-head comparative study under conditions that best mimic the intended...protein properties and provides a quick way to compare fluorescent proteins, e.g. see Chudakov et al (...is of no use. Therefore, it is more sensible to compare fluorescent proteins based on their practical brightness...promising fluorescent proteins and to determine and compare their practical brightness in a system closely ...application. In figure 2 we provide an example of the comparison of the practical brightness of two orange fluorescent...

...expressed in cells, generates much less signal than comparable fluorescent proteins. This weak emission hasn... with increased sensitivity and faster imaging compared to previous publications. They also developed ...they successfully imaged multiple subcellular compartments over several minutes using Nano-lantern fusion... fluorescence, so higher concentrations of the compound can be added to the media. Cellular esterase then...producing a constant supply of substrate. Using this compound to excite the Nano-lanterns, Takai et al. continuously... handy. This approach is especially suited to complement optogenetic tools, where light used for excitation...

...and reduce needed compensation.    Working with antibodies can get a bit complicated, but hopefully this...time-sensitive, using precious samples, or being compared to other data in a manner that demands high accuracy... reaction to understand if any of your buffer components could affect the reaction. For instance, sodium...detected in the same assay. This is one of the most complex subjects in antibody-based protocols - but thankfully...another channel's detection range. Though you can compensate for this when reading your data, you’ll want ...

...track of computation work comes with new sets of challenges. One challenge with computational analyses... of biologists are finding themselves using computational approaches to analyze large data sets (myself...RNA-seq analysis. I had never interacted with a computer at the command line, and so was really starting...especially since you’ll want to dig in to the computation, but they will serve you well as you perform ...directory: A directory is a file system in the computer and is just another name for a folder. For instance...

... do as genetic circuits become larger and more complex? Voigt: If you look at all the products of biology... on genes at different times. It requires more complex synthetic networks. Addgene: What hurdles did you... perspective and you can layer them for more complicated functions. Those things are easy with CRISPR..... There haven’t been enough genetic parts for complexity. We have characterized almost 600 terminators...of a cell. The objective is to give people the complete toolbox for taking on very large genetic engineering...just using what biology has given us. Wood is a complicated material and we can access it, but we are stuck...

...was contributed by guest blogger Alan Wong. The complexity of biological systems can hinder our attempts...simple tool that allowed us to rapidly decode the complexity? The CombiGEM-CRISPR technology was developed...of providing an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your ...mapping the combinatorial genetics that underlie complex regulation in biological systems. The CombiGEM ...new technologies to interrogate and understand complex biological systems. He can be contacted at aslw...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. PubMed ...

... and trolls. People don’t like to connect with complete strangers as it borders on my first rule of networking...’m asked a lot about requesting connections to complete strangers for informational interviews. Many have...option is in the “More…” menu. You can also follow companies and organizations to see their news and job postings...interviews.  Pro tip: You can learn a lot from the company LinkedIn page. This can include news, inclusion...It’s also a great way to keep track of past accomplishments. Follow all the rules of good resume writing...writing. Include examples of concrete accomplishments in both the technical and “plus skills” areas. Include...

...and the efficacy of your competent cells (when they get old, they lose competency). If all the above is ... be sure that the buffers and temperature are compatible between the different enzymes. Always check the...source (i.e. primer3) or provided by the major companies selling PCR reagents. Nowadays commercial polymerases...a general rule, try to use an excess of insert compared to the backbone plasmid; a starting point could...plasmid. Many vectors contain sequences that are complementary to standard sequencing primers. If not, design...

.... Addgene is now as much a tech company as it is a reagent company. I used to have to worry about this...remarkably so for a 90 person company. We were and remain ahead of most companies in creating a welcoming, ...often focus on the excitement of what we are accomplishing and what more we might do. I am grateful every...innovative thinking, and openness to team work.   Company culture Once in awhile I worry a very little ...

...specific chromosome. This image represents the composite of fluorescent measurements from red, blue, and...modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly, thereby decreasing the amount of background... labeling efficiency was similar to that of a comparable FISH method. Importantly, they did not observe...with the previously established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that...that they could evaluate the degree of chromatin compaction even for this small distance! To the authors’...CRISPRainbow generates 6 unique colors and a white composite. gRNAs with two identical hairpin binding domains...

...specific components that are key to the cloning reaction. Understanding the specific components is essential...take up your insert. But exactly how does ccdB accomplish this? Let’s start with a brief history of the...system contains ccdB. A successful insertion will completely replace ccdB with the investigator’s insert of...interest.   CcdB resistant E. coli strains complete the system But ccdB alone isn’t the only key to...protein involves poisoning of DNA-topoisomerase II complexes.” J Mol Biol. 1992 Aug 5;226(3):735-45. PubMed...

...presentation, lost in a sea of data, my mind went completely blank. After several seconds of truly panicked...honest, if my paper hadn’t been ridiculously more complex than the one before it, I don’t think I would have... panicked state, only option one would let me accomplish my goal.   As a scientist, public speaking isn't...dumpster noises were just last week!  Nerves It’s completely normal to be anxious, nervous, and/or apprehensive... in its most difficult form. You’re presenting complex data, to a group of peers who are there to provide...conferences, or even just provide quick updates during a company meeting increases visibility of your work, opens...

...based on size. Whether higher molecular weight complexes are detected will depend on the homodimer affinity...During the experiment, the solution is diluted, complicating the interpretation. Because of this, these methods...fluorescent protein is monomeric without the accompanying data to prove it is worthless. The OSER assay...and biological properties of new fusions can be compared with mEGFP or mTurquoise2, which are established...that you will be using. If you do a head-to-head comparison right, it will be valuable information for the... are engineered can be shared to simplify the comparison by others. Together, we can build a valuable ...

...us to the key question: what is the best way to compare the photostability of different fluorescent proteins...measurements for different fluorescent proteins and by comparing how the fluorescence intensity changes over time...photostability of the fluorescent proteins can be directly compared. An example of a photostability measurement of...fluorescent protein can be identified by a head-to-head comparison of several fluorescent proteins of the same spectral.... In figure 1 we provide an example of such a comparison between cyan fluorescent proteins using widefield... power, the photostability of the variants is comparable (lower panel of figure 1). To avoid changes in...

...remodeling. Chromatin is a complex of DNA and histone proteins that compacts cellular DNA into chromosomes...in the compacted region and results in a loss of gene expression. In this way, genes in compacted regions...and tightly bound DNA is to histones, the more compacted it becomes. This prevents the cellular machinery...have huge impacts in the way we study and treat complex or multifactorial diseases, enabling faster diagnosis...

...science and collaboration. The field already has comprehensive reference pages and curated databases for scientists... (CeNGEN) The CeNGEN goal is to establish a comprehensive gene expression atlas of every C.elegans  neuron...that is creating the “first virtual organism in a computer.” This project has produced various resources ...four-dimensional contrast interference (DIC) microscopy and computer imaging processing. The combination of these methods...methods provides unique opportunities to develop computational methods to better understand animal development...Hammarlund, Marc, et al. "The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System...

...significant amount of flexibility compared to a strict single-compatibility sequence like NGG. Nonetheless...NG, a rationally engineered Cas9 with NG PAM compatibility, as the name implies (Nishimasu et. al, 2018...know and love about Cas9, but with broader PAM compatibility. This alternative PAM requirement adds a significant...PAMs and is capable of editing at frequencies comparable to SpCas9. SpRY can also edit NYN PAMs, but with... Ai C, Hu J. In-depth assessment of the PAM compatibility and editing activities of Cas9 variants. Nucleic...

...blood clotting, and as we all know, a structural component of bones and teeth. It is an enzyme co-factor,..., a neuron subtype) and specific subcellular compartments for measurements. All of the sensors discussed...calcium concentrations in different subcellular compartments can vary greatly. Additionally, the calcium ...susceptible to artifacts and pH interference, and comparison between experiments can be more difficult due...GCaMP6 only) or mammalian cell expression, and compatible with constitutive or Cre-dependent expression...Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo. Neuroscience Research...