rtcb ligase (New England Biolabs)


Structured Review

Rtcb Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rtcb ligase/product/New England Biolabs
Average 93 stars, based on 1 article reviews
Price from $9.99 to $1999.99
Images
1) Product Images from "Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes"
Article Title: Small circRNAs with self-cleaving ribozymes are frequently expressed in metazoan transcriptomes
Journal: bioRxiv
doi: 10.1101/721605

Figure Legend Snippet: Circularization of Mexican axolotl self-cleaved retrozyme RNA. Purified linear monomeric RNA resulting from double self-cleavage of DRtz353 (see Figure 4C ) was run directly in a denaturing gel (lane 1), after 1h incubation with RtcB ligase in its corresponding buffer (lane 2), in RtcB buffer without protein ligase (lane 3), and in 50 mM Mg 2+ buffer (lane 4). Circular RNAs can be readily detected in lane 2 (up to 80% circularization for the 350 nt monomer) and lane 4 (3% self-circularization for the 350 nt monomer). Minor fractions of linear and circular dimeric RNAs are also indicated (lane 2).
Techniques Used: Purification, Incubation
2) Product Images from "Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts"
Article Title: Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts
Journal: Nature biotechnology
doi: 10.1038/s41587-019-0090-6

Figure Legend Snippet: Tornado expression system generates circular RNA a , Ribozymes efficiently self-cleave during transcription reactions.. The construct containing Twister P1 and Twister P3 U2A ribozymes was transcribed in vitro and quenched with urea before running on denaturing PAGE and visualizing RNA. Fully cleaved products and the side products of cleavage accumulate efficiently and rapidly after transcription. b , Fully-cleaved products of transcription in a contain appropriate ends for circularization by the endogenous ligase, RtcB. We excised the fully-cleaved RNA from a and performed an RtcB ligation reactions. RtcB treatment produces a shift in gel mobility that is not observed without ligation or with pre-treatment with T4 PNK. This shift in gel mobility suggests that the fully-cleaved RNA contains the appropriate ends for ligation. Staining of the gel with DFHBI-1T and comparison of fluorescence relative to SYBR Gold signal demonstrates that circular Broccoli is brighter than linear Broccoli. c , Twister-based ribozyme-assisted circular RNA (racRNA) expression generates significantly higher levels of circular RNA than the previous circular RNA expressing system. HEK293T cells expressed racRNA Broccoli from a variety of racRNA expression systems (see Fig. 1 ) with different combinations of 5’ and 3’ ribozymes and were compared to expression using the tricY system. Cells were treated with actinomycin D (ActD) for 6 h to observe the drop in RNA levels after new RNA synthesis was inhibited. The Twister P1 and Twister P3 U2A construct, dubbed “Tornado”, expresses high levels of Broccoli RNA that exhibit high stability, characteristic of circRNA. d , Tornado-expressed RNA is decisively circular. DNA-directed cleavage by RNase H of a linear RNA produces two bands, each of expected size given the transcript length and probe site. The identical treatment of the same sequence expressed from Tornado produces a single band similar in size to the uncleaved transcribed sample.
Techniques Used: Expressing, Construct, In Vitro, Polyacrylamide Gel Electrophoresis, Ligation, Staining, Fluorescence, Sequencing