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Pierce chemiluminescent nucleic acid detection module kit

Manufactured by Thermo Fisher Scientific
Sourced in United States

The Pierce Chemiluminescent Nucleic Acid Detection Module Kit is a laboratory equipment used for the detection and quantification of nucleic acids through chemiluminescence technology. The kit includes the necessary reagents and components to perform this analysis.

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6 protocols using pierce chemiluminescent nucleic acid detection module kit

1

Northern Blot Analysis of miR-34a

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The isolated dbRNAs were separated on a 15% urea-PAG (TBE is the running buffer) at 20 mA followed by blotting using the iBlot™ Gel Transfer Device onto a Novex™ iBlot™ DNA Transfer Stack with a nylon membrane (both Invitrogen; Thermo Fisher Scientific, Inc.). Northern blot analysis was conducted using a miR-34a specific biotinylated probe (sequence: /5Biosg/AGGGCAGTATACTTGCTGAT) after membrane was incubated with prehybridization buffer (6X SSC buffer, 10X Denhardt's solution, 0.2% SDS; MilliporeSigma) for 1 h at 42°C on rotating shaker following 3 day incubation with hybridization buffer (6X SSC buffer, 5X Denhardt's solution, 0.2% SDS; MilliporeSigma) with probe at the same conditions. The chemiluminescent signal was detected using a Pierce™ Chemiluminescent Nucleic Acid Detection Module kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's recommendations.
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2

Northern Blot Analysis of Plant mRNA

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For an early screen in T1 generation, total RNA was extracted from a single leaf of 4-wk-old plants (harvested in the middle of the day) using PureZol (Bio-Rad). Eight micrograms of total RNA were run on a 1.2% denaturing agarose gel and transferred onto the Hybond N + membrane (Amersham). Northern blotting was performed as described previously (Baudry et al. 2022 (link)) using oligonucleotide probes labeled at the 5′-end with biotin (Supplementary Table S10). The membranes were prehybridized for 1 to 2 h at 50 °C in 5 × SSC, 7% w/v SDS, 100 μg·ml−1 heparin, 20 mM Na2HPO4 (pH 7.5) and hybridized overnight in the same buffer containing 1 nM biotinylated probe. Three short washes in 3 × SSC, 5% w/v SDS, 25 mM Na2HPO4 pH 7.5 were performed at room temperature. The northern blots were developed with the Pierce Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific).
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3

Southern Blot Analysis of mtpA Disruption

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Southern blotting was carried out on a single mtpA transformant for confirmation of mtpA gene disruption. As shown in Fig. 2a, the hybridization probe was prepared by amplifying a part of the hph marker gene with primers 11–12 (Table 3) and labeled using a North2South chemiluminescent detection kit (Thermo Fisher Scientific). Genomic DNA of the transformant was digested by EcoRI and HindIII and hybridized with the probe. Southern blotting was then performed using Whatman Turboblotter transfer system (GE healthcare life sciences) and detected by using a Pierce Chemiluminescent Nucleic acid detection module Kit (Thermo scientific). The blot was imaged in Thermo ECL imager.
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4

RNA-Protein Interactome Identification

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Transcripts were generated employing the HiScribe™ T7 High Yield RNA Synthesis Kit (NEB, Frankfurt, Germany). Transcripts were biotinylated employing the Pierce™ RNA 3′ End Biotinylation Kit (NEB, Frankfurt, Germany) according to the manufacturer’s protocol. Briefly, 50 pmol of transcript was labeled by ligation with a single biotinylated nucleotide at the 3′-terminus and subsequently purified. Labeling efficiency of biotinylated RNA was determined by dot blotting whilst following the description of the Pierce™ Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Scientific, Vienna, Austria).
Proteins binding to the respective transcripts were isolated employing streptavidin magnetic beads (Thermo Scientific, Vienna, Austria) according to the manufacturer’s protocol. Briefly, beads were washed and supplemented with RNA Capture buffer. Then, 50 pmol biotin-labeled RNA was added to the beads, followed by an incubation for 30 min at RT with agitation. Protein-RNA binding buffer (Tris pH 7.5 20 mM, NaCl 50 mM, MgCl2 2 mM, Tween 0.1%(v/v)), 30% glycerol, and 20 μg of nuclear lysate were added to the beads and incubated for 60 min at 4 °C with agitation. RNA-binding protein complexes were collected and washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole) and eluted in 50 mM ammonium acetate. Proteins were separated by SDS-PAGE and subsequently silver stained.
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5

Detecting RNAs in Protein Complexes

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To detect RNAs being part of the identified protein complexes, northern hybridization assays were performed as previously described (Yoo et al., 2004). The BN‐PAGE gels were transferred (Fastblot 43B semi dry Blot; Biometra, Göttingen, Germany) onto a H‐bond nylon membrane (Amersham Hybond‐N+, GE Healthcare Life Sciences, Uppsala, Sweden), UV‐crosslinked (Stratalinker 2400, Agilent, Santa Clara, CA, USA) and prehybridized with ULTRAhyb© Ultrasensitive Hybridization Buffer (Ambion®; Life Technologies) for 1 h at 68°C. Then, 3′ biotinylated oligonucleotide probes (Supporting Information Table S1) were added and the membrane was incubated again at 68°C cooling down to 37°C for 12 h, before washing with 2× sodium saline citrate buffer (300 mM NaCl, 30 mM Na3citrate pH 7.0) at room temperature for 2 min. RNAs were detected using Pierce Chemiluminescent Nucleic Acid Detection Module Kit (ThermoFisher Scientific).
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6

Southern Blot Analysis of TDR Transformants

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The hybridization probe for the southern blot analysis of the TDR transformants was designed to consist of a single fragment containing FADO, TprC terminator, tef1 promoter and FAR sequence, originating from the previously constructed expression vector (Fig. 3a). The DNA probe was biotin labeled by using Pierce North2South Biotin Random Prime Kit (Thermo Fisher Scientific, Rockford, IL, USA) according the manufacturer’s protocol. Genomic DNA of the transformants was extracted as described above and digested with restriction enzymes BamHI, PstI, and NdeI (all purchased from Thermo Scientific, Rockford, IL, USA). Southern blotting was carried out using Whatman Turboblotter transfer system (GE Healthcare Life Sciences, Pittsburg, PA, USA) and the nucleic acid detection was carried out by using Pierce Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Rockford, IL, USA), both according to the manufacturer’s protocol.
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