...further rounds of RNAi. This process, known as “transitivity,” calls into question any claims about specificity...specificity of a given RNAi technique. While transitivity enhances the potency of RNAi, it can also increase...transgene, followed by a fluorescence readout of RNAi activity. As a plant researcher, the traditional hpRNA ...Mueller WF, Clauder-Münster S, Eberhard D, Fälth Savitski M, Grandi P, Jakob P, Michon A-M, Sun H, Tessmer... W (2019) Biological plasticity rescues target activity in CRISPR knock outs. Nat Methods 16:1087–1093...

...enzyme that has been mutated to block its catalytic activity and instead become trapped in a covalent bond ...specificity for their own ligands with minimal cross-reactivity. Other strategies include using engineered ligases...2). If you’re working with purified proteins in vitro, you can even use the same tag for multiple proteins... actin filament assembly-regulating proteins in vitro reveals complex events. a) Micrographs of a fluorescently-labeled...Vivo Covalent TMP-Tag Based on Proximity-Induced Reactivity. ACS Chemical Biology, 4(7), 547–556. https:/...

...agility as a nonprofit resource can prove to be vital during this period of constrained funding. Addgene... even more valuable as labs look to maximize productivity and efficiency with fewer resources. Fostering...community has demonstrated remarkable resilience and creativity in the face of funding challenges. By strengthening...benefit of everyone. We Want to Hear from You We invite researchers to share how these funding changes ...

...of plasmid vectors and/or rely on cumbersome in vitro synthesis of single guide RNAs (sgRNAs), are time-consuming...tracrRNA system is much more potent than the in vitro-transcribed single guide RNA (IVT sgRNA) approach...Direct comparison of Cas9 RNPs prepared with an in vitro-transcribed (IVT) sgRNA versus synthetic cr:tracrRNA...efficiently and serves as a good control for both the activity of CRISPR reagents and ICC antibody specificity...of the crRNA (aim for 40-60% GC content) crRNA activity Local chromatin structure (although we could successfully...

...especially when done in vivo. There are also sensitivity issues when attempting to characterize mutations... the need for a faster and higher-throughput in vitro approach. Using data from The Cancer Genome Atlas...them had not yet been functionally validated. In vitro assay allows first-time functional annotation of...collection to screen individual perturbations in vitro using Ba/F3 and MCF10A cellular models. Ba/F3 and...

...been removed/mutated to neutralize the nuclease activity. For strains that still carry this gene, there... purification protocol to remove endonucleases activity. For these reasons choosing the correct solution...any divalent cations like Mg2+, preventing the activity of DNase. While this is very important to preserve...temperature or at 4°C. Storing DNA: temperature and longevity You should consider a couple of more things when...

... H+ specific response cytoplasmic retention insensitivity to other ions In BRET, A mutant Renilla luciferase...mutagenized and screened for speed and voltage sensitivity. This hierarchical screen led to the generation...QuasAr2 possess mutations for improved voltage sensitivity and speed, and an endoplasmic reticulum export...construct, named CheRiff, exhibits high light sensitivity and fast kinetics. By combining both the voltage...produced luciferase is truncated and lacks enzymatic activity. The Luc plasmid (intronless) is useful for counter-screening...vector that enables protein expression in any in vitro translation system. The gene set was carefully chosen...the study of promoters with either weak or high activities (Bert, et al. 2000). To design pXPG, the authors...

... a voltage sensor AAV Historically, electrical activity in the brain was measured with implanted electrodes...reporter allows a way to monitor neuronal electrical activity through imaging. Since it’s illuminated by red...fluorescence and resting fluorescence. It is useful for activity imaging of large populations of densely labeled...fluorescence and resting fluorescence.  It is useful for activity imaging of large populations of densely-labeled...

...contributed by Aliyah Weinstein Sparse Predictive Activity through Recombinase Competition (SPARC) is a fully...788679 A destabilized GFP reporter for calcineurin activity in yeast Article contributed by Leah Schwiesow...propidium iodine (PI), allowing for calcineurin activity measurements in stressed or aging cell populations...al., 2020. Find the GFP reporter calcineurin activity here Diessl et al., Microbial Cell, 2020. https...

...this episode of Hot Plasmids! Light controls the activity of a microtubule-associated protein to regulate...quantification of base-editing and Cas9 cleavage activity Article contributed by Angela Kaczmarczyk  Listen...fluorescence is activated when APOBEC-Cas9n-UGI-mediated activity edits out a frame-shift mutation within the mCherry... utility in quantifying multiple gene editing activities. Click here to find the ACE reporter plasmid...

...motivated by a particular interest in monitoring the activity and biochemistry of living neurons in an effort... calcium-binding sites as possible for added sensitivity. As reported in Nature Methods, the FRET changes...approach he and his colleagues took to optimize sensitivities can now be applied to dozens of other published...concentrations than calcium, requiring greater sensitivity. He and his colleagues came up with a solution...

... expertise, publications, and plan for future activities; including the interest of the greater scientific...one that you can pay for, but one that you are invited to join (selected due to expertise in field). Publicity...journal editors to request to be a reviewer. Proactivity is very important in this case.  You can still...it as documentary evidence for your case – the invitation to be a peer reviewer needs to be addressed to...least 3 full-time people engaged in researcher activities. The EB-1B requires that you provide two out ...

...increase activity, enhance binding between the template and primer binding site, increase processivity, or ...prime editor sequence to improve expression and activity In addition to the numerous innovations that streamline...mutations in Cas9 previously shown to improve nuclease activity (Chen et al., 2021). In a system as complex as...R221K N394K Cas9 mutations Improve Cas9 nuclease activity ~ ~ ~ ~ ✓ ✓ ✓ Evolved reverse...

...study tested AAV infectivity after a range of pH exposures. They showed that AAV infectivity was not altered...alter the infectivity of the virus. However, there was a substantial reduction in infectivity after 3 weeks...

...WYL1 increases RspCas13d and EsCas13d cleavage activity The majority of Type VI-D loci contain accessory... by RspCas13d. RspWYL1 also increased EsCas13d activity, indicating that WYL domain-containing proteins...proteins may be broader regulators of Cas13d activity. This property makes WYL proteins an intriguing counterpart...anti-CRISPR proteins that negatively modulate the activity of Cas enzymes, some of which are also functional...

...polymerase, which has 3′→5′ exonuclease activity. T4’s polymerase activity can be thwarted if no dNTPs are available...to polymerize, allowing the slower exonuclease activity to proceed. By including only one type of dNTP... reading Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Li MZ, Elledge...

...induced and quantified by measuring the amount of radioactivity in the culture supernatant (this is indicative...degraded) and it is slow. Alkaline phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline...to generate an active enzyme. Pho8 phosphatase activity or the molecular weight shift of Pho8 from its...regardless of its localization in the cell. SEP’s pH sensitivity means Rosella will fluoresce green unless it’...

...identified seven genera in in vitro experiments, possibly because in vitro conditions did not optimally...antibiotic selection. The team tested this system in vitro and in vivo. They identified cells from 19 genera...showed that this method can be used for both in vitro and in vivo studies. Find distributed cell division...

...2020. PubMed PMID: 31937940. Detection of neural activity using a red fluorescent genetically encoded voltage...detect and output the voltage changes in neural activity when paired with appropriate fluorescent optics...combines the Cre/lox system with optogenetics. Cre activity is controlled by red light by splitting Cre and...bringing each terminus of Cre together to restore Cre activity. As soon as red light or PCB is no longer present...

... without causing high background due to cross-reactivity. When to use polyclonal antibodies One advantage...labs. Polyclonal antibodies also have a high sensitivity to the target protein due to their ability to...or fixation (IHC). Because of their increased sensitivity, polyclonal antibodies are particularly useful... epitopes, increasing the likelihood of cross-reactivity with other proteins. As with any experiment, ...