The PNK activity of the N-terminal PNK domain of ScTrl1 PNK/CPDase and BfPNK/CPDase was assayed using the synthetic oligonucleotides ribo A20 (A20) and deoxy A20 (dA20), and a mixture of the two (A20 + dA20). T4 PNK and ScTrl1 CPDase were used as positive and negative controls, respectively. The reaction mixture contained 70 mM Tris, pH 7.5, 10 mM MgCl2, and 5 mM DTT, 2 μg of enzyme, and 100 pmol A20 or dA20 or both. 55 pmol of radioactively labelled [γ32P] ATP were added to each reaction mixture, and the volume of the mixtures was adjusted to 40 μl with diethylpyrocarbonate-treated water. The samples were incubated at +37 °C, and 8-μl aliquots were removed at different time points (1, 15, 30, and 60 min). Each sample was quenched with 8 μl of 2X urea sample loading buffer (Invitrogen Novex) and heated at +90 °C for 4 min. The samples were analyzed by electrophoresis using a 15% Mini-PROTEAN TBE-Urea gel (Bio-Rad), at 150 V for 85 min, and visualized using PhosphorImager analysis.
Mini protean tbe urea gel
Manufactured by Bio-Rad
Sourced in United States
The Mini-Protean TBE-Urea Gel is a laboratory equipment used for the analysis of DNA and RNA samples. It is designed to provide efficient separation and resolution of nucleic acid molecules based on their size and charge.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Spin x centrifuge tubes, by Merck Group (2 mentions)
Next seq 50 bp single end reads, by Illumina (2 mentions)
Bioanalyzer small rna kit, by Agilent Technologies (2 mentions)
Proteinase k, by New England Biolabs (1 mentions)
Perfecta sybr green fastmix reaction mixes, by Quantabio (1 mentions)
Mouse tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Miscript primer assay, by Qiagen (1 mentions)
Quant it oligreen ssdna assay kit, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Dna loading dye, by Thermo Fisher Scientific (1 mentions)
Nylon membrane, by GE Healthcare (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
5 alexa fluor 488 fluorescent dye labelled ssdna substrate of 40 bases, by Integrated DNA Technologies (1 mentions)
E coli uracil dna glycosylase, by New England Biolabs (1 mentions)
Typhoon 9400 imager, by GE Healthcare (1 mentions)
Ribonuclease a, by Merck Group (1 mentions)
9 protocols using mini protean tbe urea gel
1
PNK Activity Assay of Trl1 and BfPNK
2
rRNA Cleavage Assay with Inhibitors
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f58rRNA (1.5 µM) was incubated with or without CL11 (10 µM), CL11-M (10 µM), and CL11-E (80 µM) in buffer R with 20 mM EDTA or Lon buffer. After 2 h incubation at room temperature, commercial RNAse A (NEB) (3.3 ng/ml) was added to the reaction. Each condition was carried out at 30 or 37°C for 1 h with aliquots withdrawn every 15 min. The reactions were stopped by adding 10 µg of Proteinase K (NEB) and incubated for 5 min at 37°C. Reaction products were analyzed on 10% Mini-PROTEAN TBE-Urea Gel (BIO-RAD). Gels were visualized by fluorescence in Typhoon FLA 9500.
3
Desiccation Stress-Induced Small RNA Profiling
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Total RNA was isolated using Trizol (Thermo Fisher Scientific) from three replicates of 30 4–7 day-old female flies after desiccation stress and under control conditions. RNA samples were treated with DNAse I (Thermo Fisher Scientific) following manufacturer’s instructions. Small RNAs were obtained by gel size selection from total RNA using 15% Mini-Protean TBE-Urea Gel (Bio-Rad, #4,566,056). The gel was run at 300 V for 50 min. Fragments between 17 and 30 nucleotides were carefully cut from the gel and were put in an Eppendorf with 250 μL NaCl 0.5 M and then were placed at 4 °C in a rotating wheel o/n. Next, the sample was transferred to Corning Costar Spin-X centrifuge tubes (Merck, CLS8162), spinned and cleaned with standard ethanol washes, and eluted in DEPEC-H2O. Quality check was performed using Bioanalyzer small RNA kit (Agilent). Libraries were sequenced using Illumina Next-Seq 50 bp single-end reads.
4
Desiccation Stress-Induced Small RNA Profiling
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Total RNA was isolated using Trizol (Thermo Fisher Scientific) from three replicates of 30 4–7 day-old female flies after desiccation stress and under control conditions. RNA samples were treated with DNAse I (Thermo Fisher Scientific) following manufacturer’s instructions. Small RNAs were obtained by gel size selection from total RNA using 15% Mini-Protean TBE-Urea Gel (Bio-Rad, #4,566,056). The gel was run at 300 V for 50 min. Fragments between 17 and 30 nucleotides were carefully cut from the gel and were put in an Eppendorf with 250 μL NaCl 0.5 M and then were placed at 4 °C in a rotating wheel o/n. Next, the sample was transferred to Corning Costar Spin-X centrifuge tubes (Merck, CLS8162), spinned and cleaned with standard ethanol washes, and eluted in DEPEC-H2O. Quality check was performed using Bioanalyzer small RNA kit (Agilent). Libraries were sequenced using Illumina Next-Seq 50 bp single-end reads.
5
DNAzyme-mediated Transcript Knockdown
6
Serum Stability of CpG-scrRNA Complexes
7
Quantifying siRNA Accumulation in Papaya Virus Infection
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Tissue cultured transgenic and non-transgenic papaya plants, virus free or 24 days after inoculation with PRSV, provided leaf RNA extracts via RNeasy Plant Mini Kit (QIAGEN) for investigation of siRNA accumulation. The integrity of RNA was checked with formaldehyde denaturing agarose gel electrophoresis27 . The conserved 544-bp sequence of the PRSV CP gene was fragmented into 11 probes averaging 50 bp in length, of which probe CP544-10 (from 451 bp to 500 bp) and probe CP544-11 (from 501 bp to 544 bp) were later confirmed to produce efficient hybridization signals (Table 1 ). The Northern blot probes were labeled with Digoxigenin-11-dUTP using DIG-High-Prime Kit. Both 15% formaldehyde denaturing agarose gels (lab made) and 15% Mini-PROTEAN TBE-Urea Gels (Bio-Rad, USA) were used to separate the small RNAs by loading approximately 60-80 μg of RNA per sample, and transferring the separated bands to nylon membranes (GE Healthcare) using the capillary siphon blot method27 . Hybridization was performed overnight at 42 °C in a hybridization incubator (Model 2000, Robbins Scientific). Immunological detection was the same as for the Southern blot, except that the containers, reagents and buffers were pre-treated with 0.1% DEPC (v:v) water to remove contaminating RNAase.
8
Fluorometric Assay for RNA Deamination
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2 μM of 5′ Alexa Fluor 488 fluorescent dye-labelled ssDNA substrate of 40 bases (5′-ATTATTATTATTATTATTATTCCCAGGATTTATTTATTTA-3′) (Integrated DNA Technologies) was incubated at 37 °C for an hour with 100 nM or 1 μM r A3G (Origene) that was either pre-treated or untreated with Ribonuclease A (Sigma Aldrich) and 2 units of E. coli uracil DNA glycosylase (New England Biolabs) in 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT and 1 mM EDTA in a volume of 10 μl.1 μl of 1 N NaOH was added to the reaction, which was then incubated at 37 °C for 15 min. After adding 1 μl of 1N HCl and 12 μl of 2X loading buffer (80% formamide, 10X TBE), the samples were incubated at 65 °C for 5 min. 10 μl of the reaction was electrophoresed on a 10% Mini-PROTEAN TBE-Urea gels (Bio-Rad). Typhoon 9400 Imager (GE Healthcare) was used to scan the gel in fluorescence mode at 488 nm.
9
RNA Degradation Kinetics Assay
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The endoribonuclease and exoribonuclease activities were measured using 6-carboxyfluorescein (6FAM)-labeled RNA substrates either in 5' or in 3' as listed in Supp Table S5. 500 nM RNA substrate were incubated with 500 nM protein in a total volume of 10 µL with a buffer containing 50 mM Tris-HCl pH 7.8, 100 mM NaCl, 8 mM MgCl 2 , 0.1 mM and 0.1 mg/mL bovine serum albumin (BSA). The reactions were carried out at 37ºC for 0-30 min for the exoribonuclease activity tests and 0-75 min for endoribonuclease activity tests.
Reactions were stopped by adding 98% formamide and 10 mM ethylene diamine tetraacetic acid (EDTA) and by incubating for 15 min at 95 ºC.
The degradation products were run on a Mini-Protean TBE-Urea gels (Bio-Rad) that was pre-run for 60 min at 200 V. The samples were run for 5 min at 50 V and then for 30 min at 200 V. Gels were visualized on a Chemidoc MP Imaging System (Bio-Rad) with 0.2 s exposure at 488 nm. The gels were then stained with ethidium bromide to reveal the molecular weight marker (ssRNA molecular weight marker, Takara). Experiments were performed in triplicate and images were quantified ImageLab (Bio-Rad, version 6.1). Decay coefficients, λ, were calculated with the formula ( ) =
, where N(t) is the amount of substrate at time t and N 0 is the initial amount. The half-lives, t 1/2 were then calculated with the formula / = ( )
.
Reactions were stopped by adding 98% formamide and 10 mM ethylene diamine tetraacetic acid (EDTA) and by incubating for 15 min at 95 ºC.
The degradation products were run on a Mini-Protean TBE-Urea gels (Bio-Rad) that was pre-run for 60 min at 200 V. The samples were run for 5 min at 50 V and then for 30 min at 200 V. Gels were visualized on a Chemidoc MP Imaging System (Bio-Rad) with 0.2 s exposure at 488 nm. The gels were then stained with ethidium bromide to reveal the molecular weight marker (ssRNA molecular weight marker, Takara). Experiments were performed in triplicate and images were quantified ImageLab (Bio-Rad, version 6.1). Decay coefficients, λ, were calculated with the formula ( ) =
, where N(t) is the amount of substrate at time t and N 0 is the initial amount. The half-lives, t 1/2 were then calculated with the formula / = ( )
.
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