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...directed evolution until they identified a mutant with significantly increased sensitivity that did not affect...compared to the standard E. coli BirA* mutant. RapID consists of this mutant BirA* and an RNA component that ...elucidate these interactions scientists have long used antibody based affinity purification and mass spectrometry...proteins, which can be a challenge for classic antibody based affinity purification/mass spectrometry ...proteins resulting in their biotinylation. The advantage of APEX is its speed, labeling neighboring proteins...calcium uniporter (Martell et al., 2012). It is important to note that the biotin-phenol reagent can be ...original APEX (Lam et al. 2014). They named this mutant APEX2.  Find the APEX2 plasmids! TurboID and minturbo...

...plasmid context). The technique is also highly relevant in this age of CRISPR; site-directed mutagenesis...facilitate the repair, the template should be resistant to Cas9 cleavage. If a plasmid contains the template...cleavage), thereby rendering the resulting construct resistant to Cas9 induced cleavage. Summary of Site Directed...and bacteria are transformed with the nuclease-resistant nicked plasmid (the PCR product). Plasmids are...interest. Gel electrophoresis diagrams of the anticipated restriction analysis results (left) and validation...figure). Template A high purity plasmid prep significantly increases the success rate of site directed ...directed mutagenesis. In addition, you may want to try different concentrations of template (e.g. 0.1-1.0 ng/...

... ready-to-use recombinant antibodies. The first collection, a set of integrin antibodies, will be available...website and blog, including our antibody guide, antibody protocols, and Antibodies 101 blog series.  Stay tuned...s website this fall. IPI antibodies will come with access to the antibody sequences, in line with the...the rest of Addgene’s recombinant antibody collection, as well as access to IPI educational resources ...resources developed to support use of their antibody tools. Addgene also offers a growing collection of educational...

...several options to quantify these events. Whole genome sequencing The only way to quantify off-target effects...there are significant editing events, serving as a proxy for total off-target effects. Quantifying off-targets...CRISPR work, this often means DNA cleavage at unanticipated sites. While off-target effects are nearly impossible...and minimize them, and ways to control for and quantify them when they can’t be avoided. What are off-target... to keep in mind, but likely will not have a significant impact on your data. You are trying to generate...better to reduce off-target effects, but it’s important to not let ‘perfection’ get in the way of ‘good...mispairing of bases. To reduce off-targets, you’ll want to select a Cas enzyme with low tolerance for mismatches...

...straightforward — simply spin down the supernatant in a centrifuge to remove any unwanted cellular debris, as you would...produced in multiple different cell types, including plants, mammals, and insects. In this post, we will compare...which we talk about below. Processing The viral supernatant is typically collected over the course of a couple...to 24 hours. Processing the virus-containing supernatant will depend on the virus type. AAV is typically...methods like ultracentrifugation, while lentivirus supernatants are centrifuged and filtered to eliminate any...than a P1 virus. Pro-tip! Centrifuge the viral supernatant in between passages to eliminate dead cells and...of production, saving about a week of work. An important note is that this is only possible because baculoviruses...

...example, mutant cells can be used in drug screens to identify genes that confer drug-resistance. Mutant cells...library plus Cas9 to generate the mutant cells, respectively (C). Mutant cells are screened (D) and "hits...post was updated on Aug 20, 2020. What genes are important in your phenotype of interest? Many scientists...not entirely known. Identifying which genes are important for a phenotype can lead to a wealth of additional...Addgene.  What makes CRISPR so special? A major advantage of CRISPR/Cas9 over previous genome editing techniques...experiment is to generate and screen a population of mutant cells to identify genes involved in a particular...library to generate a heterogeneous population of mutant cells, with each cell or set of cells containing...

...typically include a gene that is advantageous for survival, such as an antibiotic resistance gene. In contrast...expressed in and amount of recombinant protein obtained. Selectable Marker The antibiotic resistance gene allows...bacterial cells and selectively grown on antibiotic plates.  Importantly, because the bacteria from which plasmids...components that make up a plasmid and why each is important. Our “Plasmids 101” series designed to educate...naturally in archaea and eukaryotes such as yeast and plants. In nature, plasmids provide one or more functional...functional benefits to the host such as resistance to antibiotics, degradative functions, and/or virulence. All...by recruiting replication machinery proteins Antibiotic Resistance Gene Allows for selection of plasmid-containing...

...includes an antibiotic resistance cassette and you aren’t introducing an additional antibiotic resistance...selection strategy, here are a few to consider. Antibiotic selection Selection is straightforward in cases...cases where the inserted DNA includes an antibiotic resistance marker. After transformation, plate the ... cells on medium containing the corresponding antibiotic. For example, if the DNA you’re inserting into...along with your gene of interest. In these cases, antibiotic selection will select for cells that have been...contains your gene of interest. Auxotrophy Besides antibiotic selection, you can select for a successful uptake...uptake of your DNA of interest by taking advantage of bacterial characteristics such as prototrophy, the...

...sequence are important considerations for designing your gRNAs. For indels, it's not so important what location...size_unit":"px","font":"helvetica","fallback":null,"variant":null,"size":18},"button_color":{"color":"#0052a9...nom. Me love gRNAs! Comic by Maya Kostman.   Important considerations before you start an experiment ...tool among them. While this seems obvious, it is important to remember that the same is true when designing...location in the gene you target, but it is important that your gRNA sequence is designed to be highly active...from). Finally, for HDR, location is much more important because you have to target within ~30 nt of your...to the intended edit rather than some passenger variant that came along with the single cell clone (although...

...PAC-MAN (prophylactic antiviral CRISPR in human cells) is a CRISPR-Cas13 based antiviral method to degrade...protease used to cleave affinity tags from purified recombinant proteins at small and large scale.  This new ...utility of the system for use with low affinity variants of TEV. The new system has been demonstrated to...the evolution platform to generate TEV protease variants. Catalysis by TEV protease allows a transcription... The benefits of nanobodies over conventional antibodies can’t be overlooked. Nanobodies are smaller in...easier to clone, express, and purify compared to antibodies. Nanobodies are easily expressed in the cytosol...of bacteria where it can be secreted into the supernatant. Four plasmids containing different nanobodies...

...unintentionally remove connecting pathways between other distant regions. They also don’t give any clues about the...and depression.  Locomotion tests To study and quantify locomotion, scientists monitor an animal’s movement...rather than being out in the open (risky). Taking advantage of these characteristic traits, rodents can be...used since the 1970s to test the efficacy of antidepressant treatment in rodents. In this test, an animal...BDNF in the hippocampus causes a shift towards antidepressant behavior in rats in the forced swim test as...neurons (Drd1) in mPFC is responsible for the antidepressant effect. To specifically activate the Drd1 neurons... of microRNA124a in the hippocampus evokes antidepressants-like effects in rats." Psychoneuroendocrinology...

...designed 2 variants of EosFP that can withstand these conditions- mEos4a and mEos4b (12). Both variants are ...light (~400 nm), the chromophore photoconverts predominantly to the anionic form, causing increased fluorescence... random mutagenesis screening for enhanced RFP variants. PA-mCherry (E26V / A58T / K69N / L84F / N99K ...respectively. When illuminated with violet light, both variants are photoconverted to forms that emit brighter...PA-FPs. Several other irreversible PA-FP spectral variants have been discovered such as Kaede and PS-CFP2...localization, or leading to protein aggregation. Another important parameter to consider is the contrast ratio- a... are performing multicolor experiments, you will want to be sure that the light used to activate the first...

...signaling — think of a mouse with an LED brain implant or a worm wiggling back and forth under blue light...microtubule-cutting sword Microtubules are one of the most important components of the cytoskeleton. They provide cells...disrupt microtubules in cells, they have various disadvantages and limitations – for example, microtubule-destabilizing...are also available as lentiviral plasmids if you want to make a stable cell line. This design allows highly...that means this light-powered sword truly is an elegant tool. The authors demonstrated its use to probe... what if you’re interested in that other most-important component of the cytoskeleton – actin? Actin dynamics...on BcLOV4. These single-component systems take advantage of BcLOV4’s light-induced binding of the plasma...

... to Get a Faculty Position. This edition has substantial expansions on the chalk talks and Zoom interviews...emphatically deny that I have a magic formula to guarantee a reader the faculty job of his/her dreams. There...currency of scientific success is publications and grants. 2. Write succinct and easy to understand documents..., documents need to minimize jargon. Equally importantly, you really are selling yourself. A sales pitch...years. You will discuss the focus of your first grant, how you will overcome certain obstacles, alternative...not recommend putting these into slide format. You want people to focus on you and your ideas. Slides will...anyone could relate to, that people would agree is important to understand. (1-2 sentences). For example: Distinguishing...

...our Transferable Skills series, this post is not meant to be a comprehensive guide to Conflict Resolution...explaining my approach to conflict resolution, it’s important to recognize that there are different types of... poor decision making, and stalled projects. You want to identify and address these types of harmful conflict...conference poster session, you don’t necessarily want everyone who visits your poster to agree with your...also be categorized as either “personal” or “substantive”. A personality-based conflict occurs when two...a lab or work environment if left unchecked. Substantive conflict occurs when people or teams who normally...or goal. Resolving these types of conflicts is important for the individuals involved, and also for the...

...be replicated in the recipient, the only antibiotic resistant colonies isolated are those where the allelic...single crossovers, they are just spontaneous antibiotic resistant clones. But if they are blue or magenta,...hospital-associated Gram-negative bacteria become resistant to antibiotics! References Espah Borujeni, Amin, Anirudh...chromosome is to disrupt expression of a gene with an antibiotic resistance cassette. This leaves a “scar” in ... This can be accomplished by selecting for an antibiotic resistance marker from the vector (abR, Figure...efficient method to generate precise, unmarked mutants, but it frequently requires considerable optimization...mix of Type VI secreted effector toxins, toxin-antitoxin type toxins, and phage holins. They hypothesized...

...paid on fellowships instead of grants such that PIs no longer save grant money by paying relatively low...discussed during the symposium will prove incredibly important to the stability of the biological research enterprise...organizers indicate that the main question they wanted to focus on during the symposium was “How many ...some of the more detailed questions the organizers wanted to touch on in organizer David Riglar’s blog post...solutions. Keynote speaker Paula Stephan did a fantastic job of articulating many of these problems and...Stephan’s Talk (All slides can be found here). Importantly, the panel offered some great solutions to these... Talk to Your Editors I wish I had been more cognizant of this during my PhD. Submitting a publication...

...between studies is substantial (e.g, the poor performance of mRuby2 is detected by Constantini et al (2015) ...steadily increasing. Of course, you want the ‘latest and greatest’ variants with the best properties for your...dimerization tendency, as is present in EGFP. Constantini et al, (2012) developed a cell-based assay that...If homodimerization occurs (most likely in an antiparallel configuration), typical structures known as ...OSER) whorls are visible. These whorls can be quantified for individual cells, and the number of 'normal...tagRFP does not act as a true monomer in cells (Constantini, 2012), whereas it was previously concluded to..., good performance in the OSER assay is not a guarantee that using this fluorescent protein to tag your...

...are added to a cellular sample. Both antibodies have a short antibody specific DNA strand added to them....PLA where one antibody was directed toward a protein of interest but the second antibody was directed ...and, because I was studying a synaptic protein, I wanted to measure it’s levels in its normal neuronal context...designated work areas and come with health risks. Importantly, with this method it is not possible to follow...most common approach. With a good and specific antibody and the proper microscope setup we can observe...fluorescent tags into the gene locus of interest. An advantage of this alternative is the possibility of real-time...interactions in situ. In the standard PLA assay, primary antibodies directed towards two endogenous proteins of interests...

...colonies, check that the antibiotic in the plate correspond to the antibiotic resistance marker present...hopefully obtain your coveted plasmid with no substantial delays. Design, design, design Before diving ...fragments. You can generate the parts of DNA that you want to assemble together in a combination of different...difficulty amplifying your target sequence, or if you want to amplify sequences from a large genome organisms...successful and ultimately faster cloning. Remember to quantify the DNA concentration of all the samples by gel... experimental design. Full lawn of cells. The antibiotic in your plate might not work. Check the plates...E. coli  strain known to be sensitive to that antibiotic, and if the strain is growing fine, it is probably...