The reaction products were subjected to 12% denaturing polyacrylamide gel electrophoresis (dPAGE, containing 8 M urea), stained by SYBR Gold, and then imaged with a UV Gel imager from BIO-RAD (Hercules, USA). Quantitative analysis of the data was carried out by using Image Lab software. The conversion for the formation of RNA rings were calculated by the following equation:![]()
where C and L were the band intensities of ssRNA ring (C-RNA) and remaining linear ssRNA (L-RNA), respectively. The results presented here are the averages of three independent experiments. In the gel electrophoresis of the reaction of L-22d (see Fig. 1A for the sequence), an oligonucleotide complementary (DNA22) to L-22d was added to assist its staining by SYBR Gold (see Fig. S2† ), because single-stranded state of L-22d is hard to be stained.
Uv gel imager
Manufactured by Bio-Rad
Sourced in United States
The UV Gel Imager is a laboratory instrument used to visualize and document DNA or protein samples separated by gel electrophoresis. It utilizes ultraviolet (UV) light to excite fluorescent dyes within the gel, allowing the captured gel images to be analyzed and interpreted.
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3 protocols using uv gel imager
1
Quantitative Analysis of RNA Ring Formation
2
PCR Amplification and Sequencing Protocol
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Polymerase chain reaction amplification of a 1,347 bp region surrounding the target sequence was performed on selected co-transformed DNA samples using the primers listed in Supplementary Table S2 . PCR was performed using T5 direct PCR kit (plant) (Tsingke biotech, Beijing, China) in a thermal cycler with the following program: 95°C for 5 min, 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1.5 min, and 72°C for 10 min. The PCR products were then run on a 1% agarose gel, and the target bands were extracted using the Omega Gel Extraction Kit (Omega Bio-tek, GA, United States). Target gel extraction products (∼1,000 ng) were digested with 5 u MwoI restriction enzyme at 60°C for 40 min. Digested DNA was then run on a 1% agarose gel in 1 × TBE for 20 min at 180 V, then imaged on a UV gel imager (Bio-Rad Laboratories, Shanghai, China). Gel bands that appeared to be undigested were then extracted using the Omega Gel Extraction Kit (Omega Bio-tek, GA, United States). And cloned into the pMD19-T easy vector (TaKaRa, Toyoto, Japan). Five to twenty clones for each line were sequenced.
3
Quantitative Analysis of RNA Ring Synthesis
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After the enzymatic reactions, the products were diluted and subjected to the electrophoresis with 12% denaturing polyacrylamide gel containing 8 M urea (dPAGE), stained by Ultra GelRed, and then imaged with a UV Gel imager from BIO-RAD (Hercules, USA). Quantitative data were obtained by using Image Lab software. The yield and selectivity for the formation of RNA rings were calculated by the following equations:
Here, C, S and P were the band intensities of the circular RNA (C-RNA) to be synthesized, linear substrate RNA (L-RNA) and all products derived from L-RNA (consisting of C-RNA, AppRNA (adenylated intermediate) and polymeric by-products), respectively. Each experiment has been repeated at least three times, and the quantitative error of band brightness was within 10%.
Here, C, S and P were the band intensities of the circular RNA (C-RNA) to be synthesized, linear substrate RNA (L-RNA) and all products derived from L-RNA (consisting of C-RNA, AppRNA (adenylated intermediate) and polymeric by-products), respectively. Each experiment has been repeated at least three times, and the quantitative error of band brightness was within 10%.
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