rnasealert assay kit  (Integrated DNA Technologies)

Journal: Bioengineered

Article Title: Polyvinylsulfonic acid: A Low-cost RNase inhibitor for enhanced RNA preservation and cell-free protein translation

doi: 10.1080/21655979.2017.1313648

Figure Lengend Snippet: Inhibition of RNase Activity with PVSA. The relative RNase Activity of both RNase A and E. coli lysate was measured at varying concentrations of PVSA using RNaseAlert® (Ambion). The amount of PVSA required for 50% inhibition (IC 50 , inset) was determined from normalized data fit to a reciprocal semi-log response curve (n = 3, error bars represent 1 standard deviation).

Article Snippet: The assays were performed using Ambion's RNaseAlert® assay kit (IDT, IA, USA).

Techniques: Inhibition, Activity Assay, Standard Deviation

Journal: iScience

Article Title: Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics

doi: 10.1016/j.isci.2021.102996

Figure Lengend Snippet: Characterization of target-activated Cas13a trans -RNase (A–C) Varied Target RNA-1 and trans -RNA U 10 were reacted with WT (A, B) or processing mutant K1082A (C) Cas13a bound with mature (A) or both mature and precursor (B, C) crRNA-1. (Insets) Lines show linear fit to yield k app (see ). (D–F) Trans -cleavage using Cas13a bound with mature (D, E) or precursor (F) crRNA-1, activated with 10 pM Target-1, then reacted with varied trans -RNA. Lines show linear fit of time course of U 20 cleavage to determine V 0 (D) or V 0 dependence on substrates U 5 and U 10 substrates (E) or hyperbolic fit of all others (E − F) to yield kinetic constants . (G–I) Target-activated Cas13a RNP cleaves RNaseAlert, untethering quencher (Q) from fluorescein (F). Cas13a bound to mature (H, I) or precursor (I) crRNA-1 was activated by 10 pM Target RNA-1, then reacted with substrate, and fluorescence was recorded continuously (H). Lines show linear fit to determine V 0 (H, Inset) or hyperbolic fit of V 0 dependence on substrate (I) to yield kinetic constants . Data in panels (A–C and H) represent mean ± SD and in (E, F, I) represent value ±standard error (SE). See also and and and .

Article Snippet: RNase A was purchased from IDT (cat. no. 11-02-01-02).

Techniques: Mutagenesis, Fluorescence

Journal: iScience

Article Title: Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics

doi: 10.1016/j.isci.2021.102996

Figure Lengend Snippet: Cas13a cleavage of target RNA and activation of trans -RNase (A) Cas13a cleavage (yellow triangle) of dual-labeled target. Components tested on Fluorescent Target RNA-1 containing sequence targeted by crRNA-1 but not −2. (B) Target-activated Cas13a cleavage of dual-labeled trans -RNA U 10 . (C–E) Single-turnover target cleavage using 1.0 nM Fluorescent Target RNA-1 reacted with varied Cas13a bound to mature (C) or precursor (D) crRNA-1 or varied Fluorescent Target RNA-1 reacted with 5.0 nM Cas13a bound to precursor crRNA-1 (E). Lines show single-exponential fit to yield k obs . (F–G) Simultaneously recorded target and trans cleavage: RNP reacted with a mixture of Alexa 488 (F 1 )-tagged target RNA and Alexa 594 (F 2 )-tagged trans -substrate. Time courses were recorded on 5.0 nM Cas13a bound to mature or precursor crRNA-1, then reacted with a mixture of 1.0 nM Alexa 488-labeled Target RNA-1 and varied Alexa 594-labeled trans -RNA U 10 . Lines show single-exponential fit (where applicable) for target (top) or trans cleavage (bottom) to yield k obs . In panels (A–C and E) data represent mean ± standard deviation (SD). See also and .

Article Snippet: RNase A was purchased from IDT (cat. no. 11-02-01-02).

Techniques: Activation Assay, Labeling, Sequencing, Standard Deviation

Journal: iScience

Article Title: Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics

doi: 10.1016/j.isci.2021.102996

Figure Lengend Snippet:

Article Snippet: RNase A was purchased from IDT (cat. no. 11-02-01-02).

Techniques: Recombinant, Mutagenesis, Isolation, Expressing, Software