...Figure 1: Overview of using Sandwich ELISA to quantitatively measure protein concentration or antibody specificity...Figure 2: Overview of using Competitive ELISA to quantitatively measure protein concentration or antibody specificity...Figure 3: Overview of using Direct ELISA to quantitatively measure protein concentration or antibody specificity...Figure 4: Overview of using Indirect ELISA to quantitatively measure protein concentration or antibody specificity...

...after amputation. For example, the dramatic “two-headed” phenotype is observable in post-amputation planarians... experiments, as well as how to perform worm amputations. They also created an online companion resource...beta-catenin. Other gene targets do not require amputation to be visible. For example, odf2 gene knockdown...

... A major drawback of this method is the stray excitation of neighboring neurons in opsin dense regions...therapeutic methylxanthine drugs like theophylline. To facilitate future work with this goal, the Barrick lab has...N-demethylases and other required enzymes, including a glutathione S-transferase homolog from a Janthinobacterium...dark yellow fluorescent protein (YFP) with an excitation peak of 519 nm. These proteins have improved ...

...turn. The first is the passing and upcoming implementation of the Federal Labor Standards Act (FLSA) which... in salary, particularly given that this has precipitated increases to the NIH recommended pay scale –...and opportunities for us all.  For example, implementation and compliance for the FLSA changes, including...challenging, but will be aided by the development and implementation of new strategies designed to cope with current...

...consequences of that change. For example, “Mutated aspartate 123 to Lysine (D123K), lowers biosensor affinity...indicated in the “Species Other” column. Relevant Mutations Indicate the amino acid change as well as any...accept sequence files in any format. Distribution status for your plasmids: Hold for Publication or Distribute...

... NPA states that Postdoctoral scholars should have the skills and techniques needed to facilitate effective... Chair automatically as it was an understood expectation of any Vice-Chair. Following an official assessment...leader in the NPA, I had to speak as an NPA representative and speak the NPA’s stance. I had to be professional...

...approach for harvesting my AAV. I skip the PEG precipitation of the HEK293 cell culture supernatant because...pellet. My yield would be higher if I used PEG precipitation, but for my experiments, I get enough virus ...culture media, you should consider performing PEG precipitation. Instead of sonicating the cell pellet, I lyse...with this AAV lysis buffer and freeze/thaw the lystate four times in a 95% ethanol and dry ice bath to...

... However, safety requirements and expression limitations may be too constraining for some experiments,...in your experiment. Figure 2: Visual representation of common AAV serotypes used for different ...that reside outside a host cell’s genome. In this state, AAV cargo can be stably expressed in non-dividing... on the Addgene Blog Overcoming the AAV Size Limitation for CRISPR Delivery Genome-wide Screening Using...

...accounting for some of the inherent inaccuracies of computational prediction models. In addition, we provide options...most promising pegRNA designs and especially facilitates the use of prime editing in pooled settings with...models (such as PRIDICT/PRIDICT2.0) can greatly facilitate prime editing experiments, thus saving you time...Switzerland), where he developed a strong passion for computational biology. His expertise includes applying machine...

...molecular biology and bioengineering, as it has facilitated the development of a simple and scalable means... gene expression. Its catalytic domains can be mutated to inactivate the protein’s nucleolytic capability...interrogation of gene function. However, there are limitations to a system that relies on two distinct Cas9 ...possible, this strategy suffers from several limitations. Perhaps most importantly, most Cas9 orthologs...

... or C terminal of a protein and can inducibly facilitate the degradation of the tagged protein. Degrons...here are all conditional, meaning that they only facilitate degradation of their fused protein under certain...through interacting ligands. After an E3 ligase facilitates ubiquitination of the Halo-tagged protein in ...HaloTag and its PROTAC are all that is needed to facilitate degradation. HaloTag is also the degron tag with...

...using the MoClo system. The paper also gives quantitative data for regulatory elements, which has not ...example, knock out a gene with simultaneous complementation, with a knockout and expression construct on...tools, double dimerization GFPs, and circular permutation GFPs. Hopefully we will have new tools for plant...doing. They look at structure, impediments, the representation of women on committees, and how the culture...

...like guanine. This RNA editor would permit point mutations in RNA which could recapitulate or rescue known...REPAIRv1, the lab targeted 34 pathogenic G->A mutations from the ClinVar database. They successfully edited...still displays substantial off-target activity. By mutating residues in ADAR2 that interact with duplex RNA...rational mutagenesis and identified that a S375A mutation in RESCUE (named RESCUE-S) offered ~76% on-target...

...washed, and either eluted from a fixed surface or precipitated, dried, and then rehydrated. There are some ...which causes the proteins and genomic DNA to precipitate. Plasmid DNA is free in solution. Proteins and...remaining proteins and RNase. Finally, DNA is precipitated with alcohol and resuspended in TE buffer. After... the National Academy of Sciences of the United States of America, 76 2, 615-9 . https://doi.org/10.1073...

... knew that their study had significant statistical limitations, and expected to find only a few, very ...theory, if the DepMap analysis had an equal representation of every ancestry group, they should have found... researchers to ignore or miss. The lack of representation in commonly used cell lines led to an incorrect...

...systems. A putative new class 2 nuclease, Cpf1 (CRISPR from Prevotella and Francisella), was annotated in several...sophisticated genome-wide analysis. At many of its computationally predicted off-target sites, Cpf1 does not mediate...learn about CRISPR biology. Later work, like the adaptation of Cas13 to RNA targeting and RNA editing, has...

...compliance with the grantor’s requirements as well as state and federal regulations. A grant professional’s ...no surprise that the U.S. Bureau of Labor and Statistics shows that employment of grant writers is expected...consider consulting for a particular grant by another state. With the blessing of my employer, I took on my ...application writing and development, project implementation, program evaluation and grant management.  ...

...have since been initiated and today thousands of mutations in genes affecting diverse biological processes—not...deactivate specific genes are needed. Targeted mutation has been demonstrated in zebrafish using zinc-...depending on whether a gene is deactivated via mutation or morpholino. This observation unlocked novel... been tracked using microscopy and advanced computational approaches (Keller et al., 2008). This microscopy-based...

...These off-target sequences may be unintentionally mutated along with the desired target locus. The PAM sequence..., we will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants...PAM binding specificity: SpCas9 variant Mutations (relative to SpCas9) PAM sequence D1135E...human genome. Another variant, xCas9 3.7 has 7 mutations found in the REC2, REC3, and PAM interacting domains...

...determine the presence and quantity of DNA, RNA or a mutation of interest. Together SHERLOCK and DETECTR demonstrate... specificity.  SHERLOCKv2 SHERLOCK had a few limitations when it was first introduced in 2017 so the lab... pre-amplification step allowing for greater quantitation without compromising sensitivity. To detect...SHERLOCK could also detect low-frequency cancer mutations from cell-free DNA fragments as well as health-related...

...data pipeline is to perform several sequential computational tasks for which existing script(s) have been...  We started at the usual recommended place: documentation. We oriented ourselves with the basic concepts... the file name. We started small with a print statement and tested that the desired data was available...we manually inspected the output with a print statement at the end of the script or by opening the files...

...preparation by precipitation in 10% polyethylene glycol (PEG) followed by centrifugation. Precipitated pellets...i.e., genome-containing) AAV particles within representative vector preparations was quantified with electron...for viral production are propagated in the endA-mutated NEB Stable strain of E. coli. In addition, plasmids...

...TBD domains, part of the HB domain, and several mutations to improve solubility and stability (Figure 2)...through eight stages of embryonic development. Representative images of single focal planes through the middle...in natural DNA repair pathways and spontaneous mutations.  Here, the researchers were able to target multiple...abscessus either simultaneously or stepwise, achieving mutation efficiencies of up to about 9% of colonies (102...

...  ICE uses Sanger sequencing data to produce quantitative, NGS-quality analysis of CRISPR editing - enabling...For a multiplex sample, ICE includes visual representations of all detected edit types in the sample. The...complete, a new screen appears with a graphical representation of the results and a list of the analyzed samples...in your edited population and their relative representation in the edited pool. The black vertical dotted...

..., allows us to display high-quality maps and annotations without us having to invest too much of our internal...partner, connect the relevant teams, coordinate implementation, and now must focus on managing the established...I personally felt that I had to overcome some hesitation to ‘approach strangers’ and learn how to communicate...maintaining useful notes that remind me about the current state of a partnership. Every collaboration and partnership...

... constant variables that enable the correct interpretation of the effect of the independent variable you...controlled for in order to eliminate any alternative interpretation of the results. Typically, plasmid-based experiments...non-targeting control is critical to the correct interpretation of the results because it provides an important...etc, but outside constraints may impose some limitations and you should be cognizant of these when interpreting...

...sense OR antisense transcript depending on its orientation to your gene. T7lac High levels of gene expression...sense OR antisense transcript depending on its orientation to your gene. araBAD General expression Promoter... promoter with somewhat weak expression. lacIq mutation increases expression of the repressor 10x, thus...sense OR antisense transcript depending on its orientation to your gene Although this list is a great...

...post was contributed by Shravanti Suresh from Iowa State University. Since its appearance, SARS-CoV-2 has... fluorescence can be observed under either no excitation light, UV light or LED blue light.  Highlights...to our guest bloggers Shravanti Surest from Iowa State University. Shravanti Suresh is a PhD candidate ...CRISPR/Cas immune systems in the Sashital Lab, Iowa State University. She is actively interested in science...

...glutaraldehyde crosslinking: representative micrograph (C), particle orientation distribution and Fourier-...natively isolated complexes. To address these limitations, the Funabiki Lab developed a technique called...of LEAs may bias the distribution of particle orientations, this could be overcome with methods such as...

...recombination target sequences, denoted by triangles] facilitate tissue or cell type specificity. An on/off genetic...administered intravenously. To address these limitations, labs have developed techniques like CREATE (...broad):  enhanced coverage for inhibitory versus excitatory neurons Mice, rats Yes (LY6A) Chan et al...AAV.CAP-B10 CNS: balanced coverage of inhibitory and excitatory neurons Mice, marmosets Unknown Brown et... its tropism is relatively more biased toward excitatory neurons than AAV-PHP.eB (Fig. 4c, Jang et al....administration of novel engineered AAV capsids facilitates enhanced transgene expression in the macaque ...

...first generate your desired mutation in mouse ES cells, select for the mutation and inject the ES cells into... models. For simple knockout alleles or point mutations, the 8-10 month process of conventional gene targeting...PAM; if not, you can add a silent PAM or guide mutation. Designing an Easi-CRISPR conditional allele The...

...which can be switched from a default OFF to an ON state, and how you might use these in your research. Today...which can be switched from a default ON to an OFF state, as well as repressible binary systems. In recent...domain VP16. cTA binds to promoters containing putative operator sequences (CuO) (linked in groups of ...QUAS promoter is less leaky than UAS in the basal state, and co-expression of the inducer QF can increase...

...nucleotide sequences with high specificity and quantitative accuracy and even include profiling single nucleotide...Electrochemiluminescent assay for detection of extremely rare mutations based on ligase reaction and bead enrichment."...direct identification of allelic composition of mutated DNA in a mixed population of excess wild‐type DNA...probe for the detection of low-abundance point mutations." Analytical chemistry 86.1 (2013): 900-906. PubMed...

...of the population as a whole, scientists gain statistical power in their observations. However, while flow...Techniques is Best for Me?  An overview of the instrumentation A flow cytometer combines three systems to ...fusions by binding to the protein in its native state.  Antibodies-based flow cytometry can use both direct...cytometry: retrospective, fundamentals and recent instrumentation. Cytotechnology 64:109–130 . https://doi.org...

...the product either via the Sodium Acetate/isopropanol precipitation method or using a silica spin column...column available from several vendors. The precipitation method has an unfortunate side effect of messing...

... V2.0 from it's predecessor, which had a point mutation in the puromycin resistance gene that made it ...less effective in certain cell lines. This point mutation has been corrected in V2.0. pSpCas9(BB)-2A-Puro...than creating double stand breaks and may better facilitate homology directed repair. You can find all of...

...is limited by definition, the kinome is a well-annotated collection of genes that can provide a useful ...useful for imaging or gene expression assays necessitating the screening of individual plasmids separately... gRNAs that score as hits in pooled screens, facilitating more rapid follow-up. We hope the biological...

...Also in June, the G8 Science Ministers issued a statement after a meeting in London on the global science...available to others got a nine percent bump in citations for their trouble, even when all kinds of other... as much about open access as it does about the state of peer review. 8)     That same issue of Science...

...has led us to develop a searchable and sortable datatable that contains validated gRNA sequences. Validated...of the validated gRNA table You can access the datatable here. Current table columns are target gene, species... interested in. You can use the carats in the datatable headers to sort alphabetically or in ascending...

...eyestalk and transform them into a pulsating display imitating a moving caterpillar. The worm controls the snail...discovered in Drosophila melanogaster (Gilbert, 2004). Mutations in these genes are lethal, associated with defective..."Tongue replacement in a marine fish (Lutjanus guttatus) by a parasitic isopod (Crustacea: Isopoda)." ...

...plasmid sharing during COVID-19 Realizing this limitation, we worked closely with the legal partners and...As infection rates were increasing in the United States, and many universities and Technology Transfer ...Proteins, and plasmids from several labs at Florida State University became openly available. Find plasmids...

...but many throughout the world are affected by devastating diseases that affect far fewer people and therefore...provide hope to patients suffering from these devastating afflictions. Elia Di Schiavi - Spinal muscular...Nabavi is a Postdoctoral Fellow at the Vancouver Prostate Center and is affiliated with the University of...

...for scientific titles generated from the United States Standard Occupational Classification System  and...they are based on the US Occupational Employment Statistics (OES), but international Twitter users were included...female to male scientific authorship in the United States (0.428). 4. The #1 (re)tweeted domain by scientists...

...its gRNA, facilitating the live study of chromatin dynamics at a particular locus. A limitation of this ..., or how organization may be altered in disease states. dCas9 gets a colorful makeover Even without its...

...Office of Postdoctoral Affairs at UNC, called her presentation “Disequilibrium, Disillusion and The Postdoc...pretty much said it all.  She cited the oft quoted statistic that only 15% of current postdocs will be academic...Biology at Harvard Medical School, titled her presentation “How to Train Your PI”.   She focused her talk...

...until now, you’ve always had to call the United States - Cambridge, MA to be exact. Today, that changes... Plasmid sharing in Europe: Addgene’s request statistics Since 2004, Addgene has shipped more than 75,...Years of Plasmid Sharing: Interested in more statistics from Addgene's 10 years of plasmid sharing? ...

...you communicate your science, whether it’s in presentations, manuscripts, or social media. If you want ...usually ok for informal settings like lab meeting presentations. You can download larger images without the ...means the images are probably better suited for presentations and digital articles where it’s easier to give...

...techniques offer researchers different advantages and limitations.  The four main factors in which chemogenetics... The opsin ChETA, for example, is a ChR2 opsin mutated to have faster off kinetics and allow neurons to... once can result in several hours of neuronal excitation or inhibition. This sustained activation period...

...immunofluorescence. GST  Glutathione S-Transferase (GST) is a 26 kDa tag which can be isolated with glutathione resin. ...fusion protein while it is still bound to the glutathione resin, eliminating the need for an extra clean...

... making plasmid maps, but can also analyze and annotate any DNA sequence. SnapGene Viewer: The free SnapGene...donation-based plasmid analysis tool including editing, annotating, creating maps, and more. This tool is maintained...analysis tools Addgene's analyze sequence tool: Annotates features, BLASTs, aligns, digests, and translates...