The mung bean nuclease protection assay was performed exactly as described previously (11 (link)). Complementary synthetic deoxyoligonucleotides were used for hybridization and protection of specific sequences of 18S rRNA. Two thousand picomoles of synthetic deoxyoligonucleotides complementary to yeast 18S rRNA were incubated with 100 picomoles of total rRNA and digestion was carried out with mung bean nuclease (MBN Kit: M0250S, NEB) and 0.05-mg/ml RNase A (Sigma-Aldrich). Protected fragments were purified by denaturing 8-M Urea-PAGE (13%) and passive elution was carried out overnight at 4°C in 0.3-M NaAc on a shaker. Eluted fragments were then precipitated with 100% EtOH.
Mbn kit
Manufactured by New England Biolabs
The MBN Kit is a laboratory equipment product offered by New England Biolabs. It is designed to facilitate the purification and concentration of DNA or RNA samples. The core function of the MBN Kit is to provide a standardized and efficient method for sample preparation, enabling researchers to obtain high-quality nucleic acid samples for downstream applications.
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3 protocols using mbn kit
1
Mung Bean Nuclease Protection Assay for 18S rRNA
2
Quantification of Yeast 25S rRNA Modifications
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RP-HPLC analysis as well as the mung bean nuclease protection assay were performed as described previously31 (link). Synthetic deoxyoligonucleotides complementary to the specific sequence of C633–G680 of yeast 25S rRNA were used for protection by hybridization to the rRNA. After digestion by mung bean nuclease (MBN Kit: M0250S, NEB) and 0.05 mg/ml RNase A (Sigma-Aldrich) purification of protected fragments from 8 M urea-PAGE (13%) was carried out by passive elution with 0.3 M NaAc by rotation at 4 °C overnight. Precipitation of eluted rRNA fragments was done using 100% EtOH.
Isolated fragments were digested with nuclease P1 and bacterial alkaline phosphatase (Sigma Aldrich) and subsequently the nucleosides were analyzed by RP-HPLC on a Supelcosil LC-18-S HPLC column (25 cm × 4.6 cm × 5 µm) equipped with a precolumn (4.6 cm × 20 mm) at 30 °C on an Agilent 1200 HPLC system.
Isolated fragments were digested with nuclease P1 and bacterial alkaline phosphatase (Sigma Aldrich) and subsequently the nucleosides were analyzed by RP-HPLC on a Supelcosil LC-18-S HPLC column (25 cm × 4.6 cm × 5 µm) equipped with a precolumn (4.6 cm × 20 mm) at 30 °C on an Agilent 1200 HPLC system.
3
Mapping 18S rRNA by Mung Bean Nuclease
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Mung bean nuclease protection assay was performed exactly as described previously (21 (link)). Complementary synthetic deoxyoligonucleotides were used for hybridization and protection of specific sequence of 18S rRNA. Two thousand picomoles of the synthetic deoxyoligonucleotides complementary to yeast 18S rRNA were incubated with 100 pmol of total rRNA and were digested by mung bean nuclease (MBN Kit: M0250S, NEB) and 0.05 mg/ml RNase A (Sigma–Aldrich). Protected fragments were purified by denaturing 8 M urea-PAGE (13%) and were eluted passively using with 0.3 M NaAc on a shaker, overnight at 4°C. Eluted fragments were then precipitated using 100% EtOH.
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