The pDNA minipreps were incubated with Monarch RNase A (NEB, 20 mg/ml; final concentration of 0.5 mg/ml) at 56 °C for the indicated durations, followed by phenol-chloroform extraction and ethanol precipitation as previously described.19 (link) The recovered plasmid DNA was analyzed on 1% agarose gels.
Monarch rnase a
Manufactured by New England Biolabs
Sourced in Germany
Monarch® RNase A is a laboratory reagent used for the digestion and removal of RNA in various biological and molecular biology applications. It is a highly purified form of the enzyme RNase A, which catalyzes the hydrolysis of single-stranded RNA.
Automatically generated - may contain errors
4 protocols using monarch rnase a
1
Plasmid DNA Purification Protocol
2
Cell Cycle Analysis of SARS-CoV-2 Infected A549-AT Cells
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In a 6-well plate, confluent A549-AT cells were treated with 10 μM PRI-724 and the corresponding amount of DMSO for 24 h and were infected with SARS-CoV-2 (B.1.617.2 isolate, MOI 0.01) in 1% MEM. After that, cells were gently detached and fixed in ice-cold 70% ethanol for 1 h. Cells were washed three times with FACS-PBS (2% (v/v) FCS, 0.1% NaN3) and were then stained overnight at 4°C by applying PI staining buffer (50 μg/mL PI, 2% (v/v) FCS, 200 μg/mL Monarch® RNase A (New England Biolabs; Frankfurt (Main), Germany), 0.1% Igepal CA-630). Cells were analyzed for PI staining using FACSVerse™ Flow Cytometer (BD Biosciences; Mississauga, ON, Canada).
3
FTO Interactions in Glioma Cells
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Glioma cells were seeded on tissue-culture treated dishes coated with human fibronectin (Millipore) at a concentration of 10ug/mL in differentiation medium for 5 days. Cells were subsequently lysed using “mild lysis buffer” (Sigma) supplied in the Imprint RNA Immunoprecipitation Kit (Sigma) and RNA was digested with Monarch RNAseA (NEB) for 1 hour at 4C. RNA/protein complexes were IP-ed with human anti-FTO antibody (Millipore) and immunoblotted with human FTO (Millipore), Ago1 (Cell Signaling) and ILF3 (Thermo) antibodies.
4
Galapagos Ground Finch Fly Protocols
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Flies were collected in Jardín de las Opuntias on San Cristobal Island, Galápagos, Ecuador (-0.9491651°, -89.5528454°) in March-April of 2019. Adult flies were reared from pupae collected in the nests of small ground finches (Geospiza fuliginosa). When the nests were empty because nestlings died or fledged, all larvae and pupae were extracted from the nest and placed in a falcon tube with a modified lid that allowed airflow. After flies eclosed from their pupal case, they were placed in the freezer, then preserved in 95% ethanol. Preserved flies were transported to the University of Connecticut, then shipped to Northern Illinois University for further processing. DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen, Valencia, California, USA) on whole fly samples after wings and legs were removed using forceps. All samples were treated with Monarch® RNase A (New England Biolabs) to remove RNA from genomic DNA samples. Multiple samples were extracted and those with low yields of DNA or contamination were discarded from further processing. Quantification of double-stranded DNA was done using QuBit® and samples were run on a gel to assess fragmentation.
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