We narrowed to 897 results for: ANT

...site-directed mutagenesis.  Quantitative polymerase chain reaction, qPCR Quantitative polymerase chain reaction... to quantify the concentrations of multiple starting DNA templates in one sample. It is important to note...heating and cooling steps. PCR relies on heat resistant DNA polymerase from the thermophilic bacterium...aquaticus (Taq). Taq polymerase is thus a heat resistant enzyme that can withstand changes in temperature...to confirm product presence, size, and relative quantity. End point PCR is most commonly used in molecular...genotyping. It’s extremely useful but is not as quantitative as other methods of PCR. Theoretically scientists...scientists should be able to determine the quantity of DNA after a PCR reaction as the amplicon doubles every...

... you want to use optogenetics The most important question to consider is what exactly do you want to use...and components necessary to run it? And, most importantly, what are the methodological and experimental...considerations? I recently spent two months in the lab of Antonello Bonci, Director of the National Institutes of ...optogenetics for? The technique is non-trivial; you don’t want to spend months doing surgeries and making fiber...region you wish to stimulate 2) Fabrication and implantation of the optical fiber into the region of interest...any optogenetic experiment. There are numerous variants for the different types of opsins available and...of North Carolina) that offer his constructs. Importantly, control viruses (not expressing the opsin) are...

...plasmids, multiple Cas9 variants are available: wt humanized Cas9, D10A nickase mutant (Cas9n), dCas9 (transcriptional...pCAMBIA. Cas9 is plant-optimized, and plasmids are available for both monocot and dicot plants, with a choice...Cas9 toolkit for multiplex genome editing in plants.” BMC Plant Biology 14 (2014): 327. PubMed PMID: 25432517...multiplex genome editing in monocot and dicot plants.” Molecular Plant 8(8) (2015): 1274-1284. PubMed PMID: 25917172...suitable for other model systems, including E. coli, plants, Drosophila, and zebrafish!   Why use multiplexed...This increases the chance that all the edits you want to make will happen.  There are many reasons to ...with Golden Gate and Gibson assembly methods If you want to scale up the number of gRNAs in your plasmid,...

...expressing the recombinant plasmid to propagate. Any cells that contained non-recombinant vectors (re-ligated...DB3.1, which contains a mutant version of DNA gyrase (gyrA462) that is resistant to the toxic effects of...factor plasmid of E. coli, is part of a toxin-antitoxin system encoded by the ccd operon, which is responsible...another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the cell against ...the number of clones that do not contain the recombinant plasmid and therefore makes the cloning process...modern version of this older system, with added advantages that will be discussed in detail in a separate...post. Focusing on the ccdB aspect, Gateway takes advantage of the same principle that cells will not propagate...

... develop a way to quantify the take rate of mouse xenografts after cell implantation. Their goal was to...cells that represent the preexisting resistant subpopulation 2. Resistant cells among the separate replicates...ClonTracer library, Bhang et al. found that antibiotic resistant cancer cells exist prior to drug treatment...majority of the resistant populations. In fact, 88% of the barcodes within the resistant populations were...decided to do a post-doc at Novartis because she wanted first-hand experience in industry research. When...determine whether or not cancer cell populations resistant to drug treatments arise during treatment or are... outcomes (see schematic depiction below): 1. Resistant cells among the separate replicates would contain...

...travel, teaching, grant writing, etc. Also, it is unlikely that a mentor has the only relevant expertise and... phenomenon? Exploration can be important, but it is equally important to have a plan to get to mechanism...talk takes significant effort. It requires evaluation of all of the data and any recent relevant studies ... often more easily done in reverse. Assuming you want to become competitive for a Group Leader position...preparation) A history of successful funding (fellowship grants) A project with sufficient preliminary data to...your own lab and get an independent investigator grant (e.g. an R01 from the NIH, in the United States)...researcher. Depending on the position, you may also want some evidence of teaching experience. How do you...

...team, you don’t necessarily want a lot of redundant skills - you just want the necessary skills. You might...but equally important is making sure all team members understand what roles they’re meant to fill and ...equivalent of a senior thesis), I knew early on that I wanted to work with a partner. WPI’s emphasis on teamwork...year isolating pheromones from various C. elegans mutants by myself, I would have slowly gone crazy. As it...and others, we have updated our kit process substantially and have plenty of ideas for further improvements...year, or a few months, or even a few weeks. The important thing to remember is that every team should have...when I do come across one. Also, remember, one important feature that all projects have in common - and...

...Jordan Ward who is an assistant professor at the University of California, Santa Cruz. Emerging CRISPR...blogger! Jordan Ward is an assistant professor at the University of California, Santa Cruz where his group ...CRISPR/Cas9 editing strategies in C. elegans As brilliantly and irreverently illustrated in the parable of...fluorescence (GFP; Tzur et al., 2013), or rescue of mutant phenotypes (unc-119) (Dickinson et al., 2013). ...Andy Fire’s lab used oligo-mediated knock-in of a dominant mutation, known as co-conversion (Arribere et ...-CRISPR and co-conversion approaches have the advantage of being used in any genetic background, but require...repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x and 3xFLAG epitopes...

...of Adeno-Associated Viral Vectors by Oxidant-Based Disinfectants. Human Gene Therapy, 32(13–14), Article...basics of rAAV production.  Of course, you will want to make sure that your final stocks only contain...your cells, wait 2–5 days and then collect the supernatant and/or producer cells for further processing....depend on the serotype, as some rAAV serotypes are abundantly released into the culture media, while others...you can consider using unpurified AAV from the supernatant (Goodwin et al., 2020) or crude cell lysate (...  Purification To purify your rAAV from your supernatant, you can simply use polyethylene glycol (PEG)... 25 mL of 40% PEG solution to each 100 mL of supernatant (final concentration of 8%). If, however, you...

...weekend - why? Do they have an important plans? What is most important to each person? Someone has to... in the area I want to explore while also ensuring that my work gets completed? I want my company to pay...everyday What I didn’t realize was how much I was constantly negotiating, every day. As a grad student, I ...I was negotiating with my PI on how to spend my grant money (Can I attend this conference?) and with my...negotiation as a zero sum game - that is, if I get what I want, that means you don’t. In this mindset, one of us...big conference - Is one of those options more important to you and why? What are their expectations/...someone doesn’t collect their data), ask why you both want this time and what your needs actually are: Maybe...

...the acquisition of antibiotic selectable markers by the destination plasmid. Antibiotic selection allowed...suppression of background by antibiotic selection. Screening of transformants by colony PCR confirmed the...number of transformants); the greater the number of inserts, the lower the number of transformants. To compensate... by guest bloggers Becky Kucera, M.Sc. and Eric Cantor, Ph.D. from New England Biolabs. Golden gate assembly...Lambda insert at the same high frequencies as the antibiotic selection markers. The 5-, 12- and 24-assembly...larger volumes of the outgrowth containing the transformants onto the selection plate. Figure 2 shows representative...assembly was easily achievable with high levels of transformants and low backgrounds – so much so that there ...

...not to mention frustrating. One elegant solution to sidestep the antibody problem is to tag the protein ...Bressan RB, Dewari PS, Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, Bulstrode H, ...Regenerative Medicine (CRM), Edinburgh. Most commercial antibodies do not work in pull-down assays: Epitope tagging...protein complexes - from the cell-lysate using antibodies against the target protein. The captured protein...to find (or generate in-house) a pre-validated antibody for each target protein. To study 20,000 human...immunoprecipitated using a standard tag-specific antibody (Jarvik and Telmer 1998). Indeed, this method ... of interest, and a universal all-purpose “tag antibody” can be used for downstream IP/ChIP assays. Creating...

...substrates/peptides and antibodies. Example applications of this include coupling to an antibody, whereby excitation...agonists and antagonists, impacting the interpretation of experimental results. It's important to consider...required. These dyes can also be attached to an antibody to detect various targets, such as proteins, nucleic...These tags consist of a genetically encoded enzyme variant that covalently reacts with a small substrate ligand...through fluorescent labeling. There are several advantages to using these self-labeling proteins over traditional...protein of interest. Fluorescence microscopy takes advantage of the characteristics of these fluorophores to...protein function and cellular components: It's important to assess how dye-conjugation affects protein ...

...photostability, quantum yield, and expression. Create yellow (v82) and lime green (v857) variants differing ...for sfGFP to create SF-iGluSnFR. This improved variant has higher expression levels in bacteria and produces...models. Furthermore, they created three affinity variants – A184S (increased affinity for glutamate), S72A...Kaspar Podgorski’s lab have recently made iGluSnFR3 variants that are more sensitive and better localized for...predecessors. The detailed characterization of the new variants is available here. Upgrades to iGluSnFR3 To improve...kinetics, affinity, and ∆F/F0. Introduce the two variants in three different membrane-targeting flavors ...Improved photophysical properties The improved variants of iGluSnFR3 have higher expression levels and...

...' TTTV AsCpf1 RR variant 5' TYCV LbCpf1 RR variant 5' TYCV AsCpf1 RVR variant 5' TATV Campylobacter... PAM sequence. Cas9 Species/Variants and PAM Sequences Species/Variant of Cas9 PAM Sequence ...NGG SpCas9 D1135E variant 3' NGG (reduced NAG binding) SpCas9 VRER variant 3' NGCG SpCas9...SpCas9 EQR variant 3' NGAG SpCas9 VQR variant  3' NGAN or NGNG  xCas9 3' NG, GAA, or GAT SpCas9...general overview of CRISPR components, new Cas9 variants are being discovered all the time and the requirements...different systems can vary (for example, xCas9 is a variant with increased PAM flexibiliy and eSpCas9/SpCas9...protein required is the Cas9 endonuclease or a variant thereof. This individual protein has all the components...

...aims do not need to be grant aims. Each aim could be the basis of a single grant. Either way, think realistically...For example, “there is a significant need for healthy livers for transplants. Growth of new livers in .... Given this large sum, one might expect that applicants would work for months to hone a chalk talk. Yet...talk could be your plans for your first NIH R01 grant proposal or a 10-year research plan. I suggest preparing...research vision is your approach to resolving a significant knowledge gap in your field or even creating ...vague goal or a "what" question. For example, "we want a better understanding of the Z pathway" is vague...techniques that can help resolve the question, the anticipated outcomes, the alternative approaches, and what...

... histogram plots to quantify smeary plots For smeary expressions, you can quantify shifts in signal using... cytometer and see one of two results for your antibody of interest: two clear populations or a huge smear...post, I’ll walk you through how to interpret the antibody readout on a flow plot.  Note that for the flow...performed. Before you read out your plots, you’ll want to make sure you’ve also adjusted voltage and compensated...cleaner readout. A yes-no plot If you are using an antibody to mark the presence or absence of a protein, ...anything to the right of it, marking it as GFP+. Quantification for such graphs could simply be the percentage...Figure 2: (A) a smeary dot plot showing a range of antibody readouts. (B) A gate on the smeary dot plot shows...

...AAV plasmids contain several important elements, but among the most important are the inverted terminal ... ITR integrity and prevent unwanted mutations?  During propagation, mutants rapidly replicate in a growth...Next-generation sequencing is also helpful in quantifying ITR mutants since analysis of individual reads allows...Adeno-associated viruses (AAV) are an important biological tool that allow scientists to express a gene...and the ITRs are retained with the gene in a recombinant AAV (rAAV) vector (Figure 1). Everything else...outside the ITRs gets left behind.    Figure 1: Recombinant AAV vector with ITRs that flank the gene cassette...modifications and types  Since ITRs are one of the most important elements of AAV and rAAV vectors, modifications...

...disciplines, the base technology itself is constantly improving. Cas9 variants have been modified for genome editing...), revealed that these mutants do not cause off-target effects at unanticipated sites and, as predicted...specificity of the Cas9 nuclease with engineered variants: eSpCas9, SpCas9-HF1, & HypaCas9. The off-target... that CRISPR/Cas9 genome editing can result in unwanted changes at non-target sites. Means to decrease...off-target effects” have included using a Cas9 nickase variant, lowering Cas9 expression, and truncating gRNA ...substitutions throughout the groove in 32 separate Cas9 mutants. When tested for their ability to cut the EMX1 ...embryonic kidney (HEK) cells, 11 of these initial mutants retained the on-target activity of wt Cas9 while...

... whether respondents saw them as not important to very important. In addition, we also explored the barriers...save them significant amounts of time and effort and give the lab a serious competitive advantage.  Many...functionalities that are hard to imagine on paper, such as instant search through all saved data and automated writing...survey respondents indicated that cost was a significant barrier to ELN adoption. This includes financial... study, respondents were also asked about how important ease of use is for them (if they are to switch...ELN choice, with almost 80% rating it as very important. One comment reflected the desire for a flexible... category;”. The study also showed that It is important that an ELN supports all types of data, which ...

... This emphasizes the advantages of bona fide pSer-incorporation into recombinant proteins as an alternative...is a particularly important control when creating a phosphoserine-incorporation mutant for a residue that...low abundance, and thus difficult to purify in quantities necessary for study. Moreover, the ability to...usually depends on tools such as phosphospecific antibodies, which aren’t available for the majority of documented...it’s possible to generate non-phosphorylated recombinant protein and perform in vitro kinase reactions...establishment of a system in which purified, recombinant protein could be produced with site-specific ...lacked a classic cysteinyl tRNA synthetase, despite abundant incorporation of cysteine in their proteomes (...

...diseases caused by Arg-mutant proteins, but there is currently no evidence of Arg-mutant protein rescue in ...rescue multiple Arg-mutant proteins in living baker’s yeast cells, including Arg-mutant yeast homologs of... the Rare Genomics Institute to provide plasmid grants to researchers working on rare diseases through...disorders of glycosylation Paula Videira is an Assistant Professor at the Faculty of Sciences of NOVA University...other. Indeed, nearly 20% of the single missense mutant alleles that cause human disease change Arg to ...rescues the function of even a small fraction of Arg-mutant proteins could improve health outcomes for millions...that the in vitro functions of 11 different Arg-mutant enzymes could be rescued by supplementation with...

...protein. Stop light! Photoactive anti-CRISPRs halt Cas9 activity Anti-CRISPR proteins are a highly diverse...our blog post: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems.  Listen to the anti-CRISPR podcast ...Light-Dependent Anti-CRISPR Bubeck et al., 2018 CASANOVA LOV2 from A. Sativa AcrIIA4 anti-CRISPR protein...Cas9 activity. The use of nickases, prime editing, anti-CRISPR proteins, and other techniques all aim to...gene expression. Both of these techniques take advantage of light-induced dimerization to activate their...C714, generating paCas9-2. This change did not significantly alter the system’s efficiency at generating ...photoactivatable repressor (dCas9). The activity of all variants was reversible and repeatable. Since these initial...

...scientists have co-opted antibodies for tagging purposes. Different antibodies bind different protein sequences...tag their proteins of interest. Antibody based probes, such as antigen-binding fragments (Fabs) (Porter...together. By combining two optimized elements of an antibody probe, scientists can now more easily visualize...amplify and image for long periods of time.  Are antibody based probes a solution? To address these limitations...HA frankenbody To combat the issues with these antibody based probes the Stasevich lab used an alternative...alternative strategy to develop a genetically encoded antibody based probe that detects the widely used HA epitope...complementarity determining regions (CDRs, or loops) of an antibody that recognize and bind specific epitopes (Ewert...

...and provides a HiBiT-enabled framework for quantifying variant expression with a minimally disruptive tag...and endogenously relevant models, tools that increase confidence become more important. For many difficult-to-study...to growth factors, some of the most clinically-relevant proteins are also the hardest to study. One common...native protein context, but they still depend on antibody-based detection or western blotting, methods that... that are low-throughput, semi-quantitative, and provide only snapshots rather than continuous views of...tagging technology.   The bioluminescence advantage There are countless protein tags floating around...signal to gradually diminish over time; and, importantly, repeated excitation generating reactive oxygen...

...might want to pick a yeast strain best suited to it. Putting K. phaffii to work   The yeast giants - S....alternative model yeast strain and discuss why you may want to consider a yeast off the beaten path.   Yeast...have a human counterpart, enabling study of human-relevant pathways (Liu et al., 2017). Beyond human relevance...well as glucose or glycerol). K. phaffii is only distantly related to S. pombe and S. cerevisiae; evolutionarily...attention for its powerful promoter of a gene important for methanol usage. This gene promoter, AOX1, ..., was co-opted for the expression of recombinant proteins by the pharmaceutical and biotech industries...efficient at protein secretion, making harvesting recombinant proteins a cinch. This is because it doesn’t ...

...found that exo-AAVs were more resistant to neutralizing anti-AAV antibodies. This is particularly useful... to reach, and are more resistant to inactivation through anti-AAV antibodies. References György, Bence...therapeutic applications in vivo where endogenous anti-AAV antibodies often neutralize the therapeutic efficacy...spontaneously released from the producer cells into the supernatant during the production phase. However, this has...researchers discovered that these particles have some advantages over the conventionally purified AAV vectors....centrifugation steps to separate cell culture supernatant containing exo-AVVs from producer cells, and ...particles with membrane vesicles protects them from antibody recognition and inactivation (György et al., 2014...

...incompatibility with antibiotic-resistance plasmids, scientists have expelled antibiotic resistant plasmids from...encode a negative regulation system involving antisense RNAs or iterons that inhibits replication when...number in the cell falls too low (Novcik, 1987).  Antisense RNAs encoded on the plasmid either indirectly ...plasmid increases, increased amounts of these antisense RNAs inhibit plasmid replication.   Figure...incompatibility going to affect my cloning project?  If you want to clone two plasmids within the same bacterial ... uncommon, or perhaps a synthetic biologist who wants to develop a plasmid-based tool that is easy to ...Using plasmid incompatibility for therapeutic advantage Although it may seem like plasmid incompatibility...

... AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library...reach all regions of the CNS. PHP.B, a neurotropic variant of AAV9, is better than AAV9 at gaining access ...sequence of DNA and both identified lymphocyte antigen 6 complex (LY6A) as a receptor for PHP.B. Read ...PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity for C57BL/6J mice is likely...screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection. Compared...efficiency, but at a log lower dose than PHP.B.  A Ly6a variant is linked to PHP.B CNS permissiveness Both the ... labs determined LY6A, also known as stem cell antigen-1 (SCA-1), is the receptor used by PHP.B to reach...

...nature can inform us on how to create better recombinant vectors. In this blog post, we’ll define plasmid...harness those features to create broad host range recombinant vectors.  A plasmid’s host range is determined...phylogenetic groups (Jain and Srivastava 2013).  Recombinant vectors that are made from broad host range plasmids...host-independent   Building broad host range recombinant vectors Replicons, which consist of a plasmid...broad host range plasmids can be used to create recombinant vectors that can replicate in different bacteria...methanolicus, Methylosinus trichosporium, Myxococcus xanthus, Neisseria spp., Paracoccous denitrificans, Pseudomonas...Rhodospirillum spp., Shewanella spp., Thiobacillus spp., Xanthomonas campestris IncQ RSF1010, R300B, R1162 ...