...express our gene of interest. This element is responsible for initiating the transcription of your insert...eukaryotes have muliple polymerases which are each responsible for a specific subset of RNAs. To gain this specificity...transcript     Description Expression Additional considerations CMV General expression mRNA Strong mammalian...elongation factor 1 alpha Constitutive  Tends to give consistent expression regardless of cell type or physiology... used for Description Expression Additional considerations T7 in vitro transcription/ general expression...regulatable  Often paired with the temperature sensitive cI857 repressor. T3 in vitro transcription/general...

...these particularly energy-intensive systems in a more environmentally responsible manner.  Other ways to ...service items we employ dozens of pieces of energy-intensive lab equipment on a daily basis. By the beginning...100% renewable electricity! A (not so) shocking transition  In early July the Town of Watertown announced...substantially reduced in an attempt to save energy. The transition to 100% renewable electricity will allow us to...

...synthesized enzyme. Biotin protein ligase (BPL) is responsible for this highly specific post-translational protein...biotinylated. In E.coli for example, the BPL, BirA, is responsible for biotinylating only one lysine in the entire...Esmond E. Snell was able to purify the protein responsible for binding biotin, avidin. Avidin was aptly ...Kresege et al., 2004). Avidin is a glycoprotein that consists of four identical subunits that can each bind ...non-selectively label DNA and RNA. Photobiotin acetate consists of a biotin molecule attached to a photoreactive...Photobiotinylation is sometimes easier and less expensive than other enzymatic methods. Scientists have ...

...Faculty Position. This edition has substantial expansions on the chalk talks and Zoom interviews sections... and mentoring to several people that found my insights helpful. All went on to get faculty positions ...understand your proposal to give you proper consideration. 3. Present an easy to follow seminar See item...Implications for human disease" is vague. Instead, consider "A mechanistic understanding of this system will...free!) contains strategies, hints, anecdotes, and insights. After I wrote the first version of my manual,...publish this manual and earn some cash for my expensive habits of running shoes and single malt whiskies...

...being realized. There are a variety of reasons to consider using genome editing to change the genetic code...sequence and ~75 nucleotide scaffold sequence) is consistent between plants and other organisms, but the promoter...CRISPR components can be expressed stably or transiently depending on the delivery method and cell type...CRISPR components can be delivered and expressed transiently using a standard detergent, Polyethylene Glycol...GOI) into plant cells. A) The system typically consists of a Ti plasmid, into which the GOI is inserted...deliver CRISPR-Cas9 to plant cells and should be considered alternative to PEG or agrobacterium-mediated ...

...change on the opposite strand). Both are base transition editors: converting one purine base to the other...pyrimidine base to the other. Figure 1: Base transition editors catalyze purine-to-purine or pyrimidine-to-pyrimidine...BioRender.com. How does base editing work? Base transition editing requires three elements. They are, broadly... in David Liu’s lab in 2016. History of base transition editing Cytosine base editing The beginnings ...frequency of 1.2%. Although the lab did not conduct comprehensive genome-wide studies of ABE specificity, their... a similar approach. Dali Li’s lab’s A&C-BEmax consists of a fusion of both cytidine and adenosine deaminases...The other 8 base-to-base changes require Base Transition Editors, which will be discussed in another post...

... concern, or nothing to worry about all. Let’s consider several scenarios below for a CRISPR experiment...An off-target rate of 5% is far too high when considering human patients and the diseases associated with...many more. While such software typically only considers DNA sequence and doesn’t take into account chromatin...the genome to reduce off-targets, it is time to consider your Cas enzyme. Even if there are similar sequences...unnecessary for many applications, it is the only comprehensive method available. Targeted sequencing approaches...must be quantified slightly differently. For a comprehensive approach, RNA-seq would be an appropriate method...

...individual neurons, including dendritic and axonal extensions and, most importantly, any connections the neurons...neuroanatomy and neural connectivity, providing valuable insights for neuronal communication. Viral vectors and...synaptic step, thus preventing further widespread expansion of RABV. This improvement resulted in a clearer... Not all measures of transport efficiency are consistent which may be caused by the diversity of techniques...the field of AAV vectors. Conclusion An ideal comprehensive toolset for neuronal tracing and network identification... wheat germ agglutinin (WGA) as a new, highly sensitive retrograde tracer in the rat brain hippocampal...nucleus Rabies and neuronal tracing Important considerations when using AAVs Resources on Addgene.org ...

...science policy community is the one that was consistently welcoming. This bears repeating. Individuals... early career researchers who have decided to transition out of academia from graduate school or a postdoc... came to DC to do either a sabbatical year or transition away from academia as tenured professors. There...Day One Project for some example proposals and consider applying for their accelerator for some hands-... to future positions, but will also make your transition from academia to the policy world much smoother...smoother.  Action Items:  Explore this comprehensive resource developed by Dr. Kelly Singel which provides...

... This post will go over some of the general considerations you should review before using an antibody....necessarily mean that you yourself have to perform extensive validation experiments for every antibody that...data can be acceptable. However, it is your responsibility to determine what counts as adequate validation...methods. These approaches have relatively low sensitivity, which can be problematic if you are trying to... your antibody’s trustworthiness. You may also consider sharing your validation attempts by other means... test an antibody (perhaps one from Addgene!), consider sharing your results so that others in the community...

...someones) taking on these responsibilities on a team to which you belong, consider stepping up. Otherwise... function. When a select group of people are responsible for a group of specific processes, they must ...tasks done. At Addgene, the Scientist Team is responsible for guiding new deposits into the repository,...roles they’re meant to fill and what their responsibilities are. That’s a subtle but important distinction...- and really can’t - be done alone. So, if you consider yourself part of the scientific community, get...

...aspects of the subject you're writing and then condensing that and making it accessible to students.” Making...for example, cutting open a heart to look at the inside. We do a lot of interactives and videos, as well...specimens.” And since the eScience warehouse is onsite, Caitlin can visit the operations team to make ...curriculum, but has taken on other new and exciting responsibilities.“I’d say maybe 25% of my day is still what...lab: write!” Caitlin did this by editing 15+ comprehensive exam proposals, a few PhD theses, and occasionally...

...multiple high-profile publications. My classmates consisted of graduate students, post-grads, and early career...topic by the moderators. The assignments, which consisted of reading that class’s section of a manuscript...given the opportunity to sign up for a writing intensive weekend in Washington D.C. at the ASM headquarters...Four ways to improve your next manuscript 1. Be consistent with your figures The figures of a manuscript...and they should also be easily understood and consistent in design and layout. 2. Be concise in your writing...

...will discuss some of the features you may want to consider as you make your choice. The first and most important...selection marker or antibiotic resistance. Next, consider the expression level of your construct: would ...pcDNA3-EGFP). If you can get away with generating transiently transfected cells, you will not need a mammalian... lentiviral or a retroviral backbone. This is considered to be a more robust expression system and has...are several types of yeast expression vectors. Consider your applications and experimental design. You...

...society 6)   Consider participating in a future SciFund Challenge. While the goal is ostensibly to get some...magazines or newspapers or other places online, and consider how those stories are being told. Twitter can ...science blogs written by practicing scientists. 3)   Consider starting up your own twitter feed or talking about... both your plasmids and your research stories. Consider practicing communicating your research by guest...

...know all about antibiotic resistance genes, let’s consider another basic element of any plasmid: the origin... intend to use, and whether or not you need to consider your plasmid's compatibility with one or more ...copy number varies. See below for additional considerations. *Requires pir gene for replication (reference... external factors contribute as well. These considerations are especially useful to keep in mind if you...affect copy number. Some ori's are temperature sensitive; others ori's can be "tricked" into amplifying...

...University of North Carolina (plasmids at Addgene). It consists of two domains, LOV2 and Zdk1, where Zdk1 binds...attract other proteins involved in their growth and extension. Now, they split EB1 into two halves (Fig. 1):...RNA localization in mammalian cells. These tools consist of cobalamin-binding riboswitches that bind to ...single base-editors has resulted in the rapid expansion of the genome editing toolkit. However, a challenge...quantitative editing assay. To help accelerate the expansion of genome editing tools, the Reuben Harris lab...

...The light intensity used to activate the PA-FP should also be considered. If the light intensity needed ...your experiment, there are several parameters to consider, such as brightness, photostability, pH stability...protein aggregation. Another important parameter to consider is the contrast ratio- a comparison of PA-FP brightness...microscope will have it’s own quirks (level of zoom, intensity, possible excitation lasers) it is also important...

... donor and acceptor fluorophores, it’s best to consider these cases separately when talking about the ...the tools that are available. Let’s consider the donor first where labeling specificity is most important...AnapRS Mammalian Cells Note: This is not an extensive list Acceptor labeling: Sometimes it's okay to... (i.e. >100 Å). Researchers have developed an extensive array of chemical labeling strategies suitable...understanding of your protein that can lead to novel insights about its biology and its behavior. See the table...covalent labeling of recombinant protein molecules inside live cells." Science281.5374 (1998): 269-272. PubMed...

...conformational changes lead to an increase in fluorescence intensity from the inserted GFP. After characterizing iGluSnFR...by glutamate, and tweaking it to improve its responsiveness, they showed that iGluSnFR could detect neuronal...and produces stronger fluorescent signals upon sensing glutamate in mouse and ferret models. Furthermore...recently made iGluSnFR3 variants that are more sensitive and better localized for synaptic glutamate response... and produce stronger fluorescent signals upon sensing glutamate using both 1-photon and 2-photon imaging...