Total RNA was isolated from the RNAi cells in which RNAi was not induced or induced for 48 h, or from PF 3a RNAi-CrPV IRES and PF 29-13 cells, using the TRIzol Reagent (Life Technologies) according to the manufacturer's instructions. Twenty micrograms of RNA from each sample was resolved on a formaldehyde-containing 1.2% agarose gel and then transferred to a Hybond N+ membrane (GE Amersham Biosciences). The RNA samples from RNAi cells were hybridized with the DIG-labeled probe specific to TbIF3a or TbIF3f. The RNAs from PF 3a RNAi-CrPV IRES and PF 29-13 cells were hybridized with the labeled probe specific to Rluc or Fluc (Fig. 5 A), and then stripped and rehybridized with the probe specific to α-tubulin, which was used as a control. Probe labeling and signal detection were conducted with DIG High Primer DNA Labeling and Detection Starter Kit II according to the manufacturer's instructions (Roche Diagnostics). The rRNAs were observed through ethidium bromide (EtBr) staining.
Dig high primer dna labeling and detection starter kit 2
Manufactured by Roche
Sourced in Switzerland, China
The DIG High Prime DNA Labeling and Detection Starter Kit II is a laboratory equipment product designed for the labeling and detection of DNA probes. It provides the necessary components to label DNA probes with the hapten digoxigenin (DIG) and subsequently detect the labeled probes in various applications such as Southern blotting, in situ hybridization, and colony/plaque screening.
Automatically generated - may contain errors
Lab products found in correlation
Chef mapper xa pfge system, by Bio-Rad (2 mentions)
S1 nuclease, by Takara Bio (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Positively charged nylon membrane, by Merck Group (1 mentions)
Plant genomic dna kit, by Tiangen Biotech (1 mentions)
N magaprobe nylon transfer membranes, by Dingguo (1 mentions)
Hybond nylon membrane, by GE Healthcare (1 mentions)
G 25 sephadex columns, by GE Healthcare (1 mentions)
Dig detection kit, by Roche (1 mentions)
8 protocols using dig high primer dna labeling and detection starter kit 2
1
Northern Blot Analysis of RNA Expression
2
Plasmid Detection and Characterization
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Bacterial genomic DNA was prepared in agarose plugs and digested with S1 nuclease (Takara). The linearized plasmids and partially digested genomic DNA were separated through the CHEF-Mapper XA PFGE system (Bio-Rad). The DNA fragments were stained with ethidium bromide (EtBr), transferred to a Hybond N+ membrane (GE Amersham Biosciences) and hybridized with a DIG-labeled probe specific to blaNDM (Rasheed et al., 2013 (link)). Probe labeling and signal detection were carried out with DIG high primer DNA labeling and detection starter kit II according to the manufacturer's instructions (Roche Diagnostics).
3
Generation and Analysis of Transgenic Cotton Lines
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The transgenic GhACS2/6-OE lines were created as previously reported [25 (link), 26 (link)]. Briefly, the open reading frame of GhACS2 or GhACS6 was inserted into the vector pK7WG2 with CaMV 35S promoter, respectively, and introduced into Agrobacterium tumefaciens strain EHA105, and then used to infect the hypocotyl of YZ1 seedings, respectively. The positive plants were screened on 1/2 MS medium containing 50 μg/mL kanamycin, until T3 lines for research analysis were obtained.
Genomic DNA was extracted from young leaves of YZ1 using a plant genomic DNA kit (TIANGEN Biotech, Beijing, China). For Southern blotting, 20 μg of genomic DNA was digested with the restriction enzyme HindIII overnight, separated on a 0.8% agarose gels by electrophoresis and transferred onto a positively charged nylon membrane (Millipore, Billerica, MA, USA). The nylon membrane was hybridized with DIG-11-dUTP-labeled fragments at 45°C. A DIG High Primer DNA Labeling and Detection Starter kit II (Roche, Basel, Switzerland) was used for labeling and hybridization in accordance with the manufacturer’s protocol. Homozygous seedlings from single insertions and high expression lines were selected for the following studies.
Genomic DNA was extracted from young leaves of YZ1 using a plant genomic DNA kit (TIANGEN Biotech, Beijing, China). For Southern blotting, 20 μg of genomic DNA was digested with the restriction enzyme HindIII overnight, separated on a 0.8% agarose gels by electrophoresis and transferred onto a positively charged nylon membrane (Millipore, Billerica, MA, USA). The nylon membrane was hybridized with DIG-11-dUTP-labeled fragments at 45°C. A DIG High Primer DNA Labeling and Detection Starter kit II (Roche, Basel, Switzerland) was used for labeling and hybridization in accordance with the manufacturer’s protocol. Homozygous seedlings from single insertions and high expression lines were selected for the following studies.
4
Detecting Replicative and Encapsidated AAV DNA
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For Hirt DNA samples, ∼3 μg of the extracted DNA was digested with DpnI overnight at 37°C. The input plasmid DNA was digested and newly replicated AAV DNA was not affected. (For extracted encapsidated vector DNA samples, this step can be skipped.) DNA samples were separated on a 0.8% native or alkaline agarose gel at 4 V/cm for 5 h at 4°C. The gel was removed from the electrophoresis tank and incubated for 30 min in 0.25 M HCl denaturing solution on a platform shaker at ∼40 rpm. The gel was then rinsed twice in double-distilled H2O. After the rinse, the gel was incubated for 30 min in 0.4 M NaOH neutralizing solution on a platform shaker at ∼40 rpm, and then the assembly of the transfer setup could be undertaken. Transfer the DNA in the gel to the nylon membrane by the capillary method for overnight. The setup was disassembled and the membrane was removed carefully. The subsequent DNA fixation, hybridization, immunological detection steps were carried out according to the protocol provided by DIG High Primer DNA Labeling and Detection Starter Kit II (catalog no. 11585614910, Roche). The DIG-labeled ITR probe was applied to detect replicative or encapsidated AAV DNA. The DNA intensity was analyzed by Image Lab software.
5
Bacterial Plasmid Identification via PFGE
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Bacterial genomic DNA was prepared in agarose plugs and digested with S1 nuclease (Takara). The linearized plasmids and partially digested genomic DNA were separated through the CHEF-Mapper XA PFGE system (Bio-Rad). The DNA fragments were stained with ethidium bromide (EtBr), transferred to a Hybond N+ membrane (GE Amersham Biosciences) and hybridized with the DIG-labeled probe specific to blaNDM-1 or blaOXA-58. Probe labeling and signal detection were carried out with DIG high primer DNA labeling and detection starter kit II according to the manufacturer's instructions (Roche Diagnostics).
6
Genomic DNA Extraction and Analysis
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Yeast strains were cultivated in YPD liquid medium at 30 °C overnight, and the genomic DNA was extracted as previously described34 (link). Appropriate restriction enzymes as denoted in the legends were utilized to digest the genomic DNA. The digested DNA was then subjected to separation on 0.7% agarose gels and transferred to N+-Magaprobe nylon transfer membranes following the manufacturer’s instructions (Dingguo, Beijing, China). The probe (as indicated in each figure) was labelled with digoxigenin by following the manufacturer’s instructions for the DIG High Primer DNA Labeling and Detection Starter Kit II (Roche China, Shanghai, China).
7
Northern Blot Analysis of GFP mRNA
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RNA (‘northern’) blot analysis of GFP mRNA was performed as described previously (Du et al., 2014a (link)). Total RNA was extracted from agroinfiltrated leaves using TRIzol reagent (Invitrogen) according to the manufacturer’s instruction. Five micrograms of each total RNA sample was separated on 1.5% agarose gel containing 7% formaldehyde, and transferred onto Hybond+nylon membrane (GE). GFP mRNA was detected by the digoxigenin (DIG)-labeled DNA probes that were prepared using the DIG high primer DNA labeling and detection starter kit II (Roche) as recommended by the manufacturer, and cleaned up using G25 Sephadex columns (GE). DIG-labeled probes were detected using a chemiluminescence-based DIG detection kit (Roche) according to the manufacturer’s instructions.
8
Genomic DNA extraction and Southern blot analysis
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Overnight cultures (16 h) were harvested from the YPD liquid medium by centrifugation, and the genomic DNA was extracted and digested with EcoRV and SalI. Probe was amplified from the wild-type genome using primer pair Xrn1-up-F/Xrn1-down-R and labelled with digoxigenin. A Southern blot procedure followed the protocol of DIG High Primer DNA Labeling and Detection Starter Kit II (Roche, Mannheim, Germany) as previously described [24 (link)].
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