E. coli strains were grown in LB at 37°C or at 30°C to exponential phase, diluted to an OD600 of 0.01 in fresh KB and then grown to mid exponential phase (OD600 ~0.4). Rifampicin was added to a final concentration of 200 μg/ml to stop transcription initiation. Samples were harvested at 0, 1, 2, 4, 8, and 16 min following rifampicin addition and immediately mixed with ice-cold 1/8 sample volume of stop solution (10% phenol: 90% ethanol [v/v]). Total cellular RNA was isolated using hot phenol-chloroform extraction followed by ethanol precipitation. Genomic DNA was digested by treating 20 μg of nucleic acids with 2 U of Turbo DNase (Roche), and RNA was purified from these reactions with phenol-chloroform extraction followed by ethanol precipitation. Spot 42 or rpsT RNA levels were detected by Northern blotting and normalized to the 5S rRNA levels. Semilog decay curves were determined using Prism (GraphPad).
Turbo dnase
Manufactured by Roche
Turbo DNase is a lab equipment product designed for the rapid and efficient digestion of DNA. It is a highly active recombinant DNase enzyme that effectively removes DNA from samples, facilitating downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
2720 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions)
Turbofect, by Thermo Fisher Scientific (1 mentions)
Decitabine, by Merck Group (1 mentions)
Proteinase k, by Roche (1 mentions)
Megascript t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Rnase t1, by Roche (1 mentions)
5 protocols using turbo dnase
1
Measuring E. coli RNA Stability
2
Transcriptional Profiling of HPV-Positive Tumor Cells
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Total RNA was isolated from HPV DNA-positive tumor cell lines using the PureLinkTM RNA Mini kit (Thermo Fisher Scientific, Inc.). RNA purity and quantity were evaluated spectrophotometrically. Total RNA was treated with Turbo DNase (Roche Diagnostics), and complementary DNA was synthesized using random hexamers according to the SuperScript™ II Reverse Transcriptase protocol (Thermo Fisher Scientific, Inc.). To verify E6/E7 transcript expression, PCR was conducted with AmpliTaq Gold® DNA Polymerase (Thermo Fisher Scientific, Inc.) on a 2720 Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific, Inc.), using pairs of primers designed based on the conserved sequence regions of the HPV16 and HPV18 genotypes (Table I ). HT3 cells served as HPV-negative controls.
3
Quantitative RT-PCR Analysis of HPV16 Splice Isoforms
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RT-PCR was performed as described before (12 (link)). Briefly, total RNA was treated with Turbo DNase (Roche), followed by RT with a random hexamer primer and Moloney murine leukemia virus (MuLV) reverse transcriptase (Life Technologies). PCR was subsequently performed with AmpliTaq (Life Technologies). For real-time RT-PCR, a TaqMan probe with 5′-6-carboxyfluorecein (FAM) and 3′-carboxytetramethylrhodamine (TAMRA) and oligonucleotide primers were designed from the conserved sequence regions among HPV16 subtypes (Table S3 ). Total RNA (400 ng) from cervical cancer cells or tissues was used for each real-time RT-PCR, and relative copy numbers of each splice isoform were determined from the cycle threshold (CT) value of the isoform RNA against a standard curve created from the corresponding cDNA plasmids.
4
Mapping MDA5 Interactome by FLASH
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pMYS-MDA5-FHBH was generated by subcloning the human MDA5 DNA sequence and the FHBH tag(3FLAG-6His-Biotin-6His) at the C-terminus of MDA5 into pMYS-IRES-GFP. HEK293T cells were transfected with pMYS-MDA5-FHBH or pMYS-GFP-FHBH with TurboFect (R0531, Thermo Fisher Scientific). The next day, cells were either irradiated (10 Gy) and collected 6 h later or treated with 1 μM decitabine (A3656, Sigma-Aldrich) for 72 h (exchanged daily). FLASH was performed as previously described28 (link),83 (link) and two replicates for each condition were sequenced using the NextSeq 500 system. The only difference with the published protocol is that washes after the streptavidin pull-down were performed with 0.1% SDS, 1 M NaCl, 0.5% LiDS, 0.5 M LiCl and 1% SDS, 0.5 M LiCl. For FLASH qPCR in HEK293 and OP9 cells, 100 µl input was taken before the first pull-down. After the streptavidin pull-down, the beads were treated with TurboDNase for 2 h for the input and 1 h followed by proteinase K (03115836001, Roche). Spike-in luciferase control RNA (15 pg μl) (L4561, Promega) was added and RNA isolation, cDNA and qPCR were performed as described above.
5
Synthesizing and Delivering dsRNA in Planarians
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Double-stranded RNA (dsRNA) was synthesized based on a previous report (Rouhana et al., 2013) .
A DNA fragment containing the target gene sequence with the T7 promoter at both ends was used as the template for dsRNA synthesis. The synthesis was conducted with a Megascript T7 transcription kit (Thermo Fisher Scientific) following the manufacturer's protocol. The synthesized dsRNA was treated with TURBO DNase and RNase T1 (Roche) for 1 h at 37°C to remove DNA template and single-stranded RNA. The dsRNA was precipitated with LiCl and dissolved in water at 250 ng/l. Thirty planarians were fed a mixture of 25 L of 50% chicken liver homogenate (w/v), 5 L of 2% agarose (w/v), and 10 L of dsRNA solution, once daily for 3 h. The mixture was divided into small aliquots and frozen at -30°C before feeding. The feeding was conducted on four successive days and on the day of 1 week after the first feeding. Control animals were fed dsRNA of enhanced green fluorescent protein (EGFP).
A DNA fragment containing the target gene sequence with the T7 promoter at both ends was used as the template for dsRNA synthesis. The synthesis was conducted with a Megascript T7 transcription kit (Thermo Fisher Scientific) following the manufacturer's protocol. The synthesized dsRNA was treated with TURBO DNase and RNase T1 (Roche) for 1 h at 37°C to remove DNA template and single-stranded RNA. The dsRNA was precipitated with LiCl and dissolved in water at 250 ng/l. Thirty planarians were fed a mixture of 25 L of 50% chicken liver homogenate (w/v), 5 L of 2% agarose (w/v), and 10 L of dsRNA solution, once daily for 3 h. The mixture was divided into small aliquots and frozen at -30°C before feeding. The feeding was conducted on four successive days and on the day of 1 week after the first feeding. Control animals were fed dsRNA of enhanced green fluorescent protein (EGFP).
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