In order to determine the number of transgene integration events in the 14 transgenic lines, Southern blot hybridisation was carried out using DIG-High Prime DNA Labelling and Detection Starter Kit II (ThermoFisher Scientific) according to the manufacturer's instructions. Twenty micrograms of DNA from each line was digested overnight with HindIII (15053–15059) which cuts once in the cassette, and EcoRI, restriction enzymes (ThermoFisher Scientific) and separated using gel electrophoresis, in a 1% agarose gel in 1X TAE. The DNA was then transferred to positively charged nylon Hybond-N + membrane (Amersham), pre-hybridised at 38 °C, and hybridised at 64 °C overnight with a DIG-labelled Hyg gene probe, which was labelled using the DIG-High Prime DNA Labelling and Detection Starter Kit II (Roche). Pre-hybridization (3 h) and hybridization (overnight) were carried out using DIG EasyHyb buffer (ThermoFisher Scientific). Any unbound probe was washed from the membrane by sequential washing with buffers 2xSDS, 0.1% SDS twice, and 0.1xSDS, 0.1% SDS buffer twice (incubated at 40 °C). Signal detection (CDP star) was performed following DIG-High Prime DNA Labelling, and the Detection Starter Kit II (Roche) protocol. Results were visualised using GelDoc XR+ (Biorad) after 10 min.
Detection starter kit 2
Manufactured by Roche
Sourced in Germany, Switzerland
The Detection Starter Kit II is a laboratory equipment product from Roche. It is designed for the detection and analysis of various biological samples. The core function of this kit is to provide the necessary tools and reagents for researchers to conduct preliminary analyses and prepare samples for further investigation. The details of its intended use and specific applications are not included in this factual description.
Automatically generated - may contain errors
Lab products found in correlation
Pcr dig probe synthesis kit, by Roche (3 mentions)
Hybond n membrane, by Cytiva (2 mentions)
Hybond n membrane, by GE Healthcare (2 mentions)
Dig high prime dna labelling and detection starter kit 2, by Roche (1 mentions)
Hybond n nylon membrane, by Cytiva (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Genome walking kit, by Takara Bio (1 mentions)
Peasy blunt vector, by Transgene (1 mentions)
X ray film, by AGFA HealthCare (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Uv transillumination, by Bio-Rad (1 mentions)
Chef mapper electrophoresis system, by Bio-Rad (1 mentions)
Nylon membrane, by Cytiva (1 mentions)
Semi dry transfer system, by Bio-Rad (1 mentions)
Hybridization buffer, by Roche (1 mentions)
Enhanced sensitive ish detection kit 1, by Boster Bio (1 mentions)
Dm2000 microscope, by Leica (1 mentions)
Chef mapper xa pfge system, by Bio-Rad (1 mentions)
S1 nuclease, by Takara Bio (1 mentions)
Nylon membrane, by Roche (1 mentions)
17 protocols using detection starter kit 2
1
Transgene Integration Analysis via Southern Blot
2
Isolation and Identification of T-DNA Insertion Sites
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TAIL‐PCR (Liu et al., 1995 ) was used to isolate regions flanking the T‐DNA insertion with Genome Walking Kit (TaKaRa, Tokyo, Japan). PCR‐amplified fragments were cloned into the pEASY‐Blunt vector (TransGen Biotech, China) and then sequenced (Biomed Biotech, Beijing, China). For Southern blotting, about 10 μg genomic DNA from wild‐type and spl35 mutant were digested with HindIII overnight, and digested products were separated in 1% agarose gels and then transferred onto a Hybond‐N+ membrane (Amersham Pharmacia, Biotech, Buckinghamshire, UK). A 481‐bp fragment of the hygromycin phosphotransferase gene amplified from the pCUbi1390 vector by the primer pair Hyr‐F/R was DIG‐labelled and used as a hybridization probe. Southern blotting was performed with DIG high prime DNA labelling and a detection starter kit II according to the manufacturer's instructions (Roche, Mannheim, Germany). Primers used for TAIL‐PCR and Southern blotting are listed in Table S1 .
3
LbAmastin Expression in Leishmania
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Roughly 3.6 × 108 promastigotes were used to isolate RNA by TRI reagent method (Sigma Aldrich, St. Louis, MO, USA) following manufacturer’s instructions. About 8 μg of total RNA were loaded onto 1.5% (w/v) low electroendosmosis agarose/MOPS/formaldehyde gels and, after electrophoretic separation, transferred onto nylon membranes. Probes used in these studies were prepared and labeled with digoxigenin during PCR amplification using specific primers for each gene and the PCR DIG Probe Synthesis kit (Roche, Mannheim, Germany). A probe of LbAmastin coding region was obtained based on the LbrM.20.4320 gene, using the LbAmasFw (5′-ATG AAG CGG AGT GTT CCC ATT C-3′) and LbAmasRv (5′-CTA CTC CTC CTC CTC CTT TTG TGT G-3′) primers. Hybridization and immunological detection were performed using the Detection Starter kit II (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Finally, membranes were exposed on X-Ray film (AGFA, Mortsel, Belgium).
4
Plasmid Characterization of Salmonella Derby
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Salmonella Derby strains Sa64 and its corresponding transconjugants were subjected to S1-PFGE to obtain the length of the plasmids. Briefly, S1-nuclease was used to digest agarose embedded DNA at 37°C for 15 min. The restriction bands were dispersed by using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA, United States) with 2.16 to 63.8 S pulse times in 0.5 Tris–borate-EDTA buffer at 14°C. DNA fingerprinting of H9812 was used as DNA dimension marker. The gels were subjected to GelRed staining, and DNA bands were imaged with UV transillumination (Bio-Rad). Southern bolt hybridization was conducted according to the manufacturer’s directions of the Detection Starter Kit II (Roche Diagnostics), using the digoxigenin-labeled qnrS gene probe.
5
Northern Blot Analysis of miRNA
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Total RNAs were loaded to the wells of a denaturing 15% polyacrylamide gel using 1× TBE buffer (90 mM Tris-boric acid, 2 mM ethylenediaminetetraacetic acid [EDTA] [pH 8.0]). Following electrophoresis, RNA was transferred to a nylon membrane (Amersham Biosciences, UK). The membrane was hybridized overnight at 55°C with a digoxin (DIG)-labeled DNA probe (miR-17, 5′-CTA CCT GCA CTG TAA GCA CTT TG-3′; U6, 5′-GGG CCA TGC TAA TCT TCT CTG TAT CGT T-3′) after UV crosslinking. The membrane was subsequently detected using Detection Starter Kit II (Roche, Germany) and DIG High Prime DNA Labeling.
6
One-step Growth Experiment for Bacteria-Phage Co-culture
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Bacteria-phage co-cultures were prepared by one-step growth experiment66 (link). Strain 1–9, 1–9/pLLY1101, or PAK-AR2 was incubated with the phage K5 at an MOI of 1. Multiple 20-ml cultures were taken at 20 min intervals. Total genomic DNA was extracted from the collected cells according to the method described previously67 . DNA samples were digested with the restriction endonuclease EcoRV for southern blot analysis. Primers were designed to synthesize the probe corresponding to region from 2854 to 3373 bp of the K5 genome (Supplementary Table 3 ). The genomic DNA of PAK-AR2 and the phage K5 was used as negative and positive controls, respectively. DNA probe synthesis and DNA hybridization were conducted using the Detection Starter Kit II (Roche) according to the manufacturer’s instruction.
7
Northern blot detection of CWMV RNA
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Northern blot assays were performed as previously described (Yang et al., 2016 ). Briefly, total RNA (c.5 µg per sample) was separated in a 2% formaldehyde agarose gel through electrophoresis (60 V for 1.5 hr). The separated RNA bands were transferred onto a Hybond‐N+ membrane (Amersham Biosciences) followed by a 10 min crosslink under a UV light. The blot was hybridized with a digoxigenin‐labelled probe specific for the 3ʹ end of CWMV genomic RNAs. The probe was produced using a Detection Starter Kit II following the manufacturer's instructions (Roche).
8
Identification of mcr-1 plasmids
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Southern blotting was performed using a semidry transfer system (Bio-Rad) and the mcr-1-containing plasmids were identified by hybridization with Dig-labeled mcr-1-specific probe generated by the PCR DIG Probe Synthesis Kit, and Detection Starter Kit II (Roche Applied Sciences, Mannheim, Germany) [21 (link)].
9
Kinetics of SNJ1 Virus Infection
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CJ7-F cells possessing pWHU3808 and pFJ6-H were incubated with the SNJ1 virus at an MOI of 5. Multiple aliquots of 10 mL of culture were collected at different time points (1, 5, 10, 15, 30 min and 1, 2, 3, 4, 5 and 6 h) post infection. In addition, samples of the cultures prior to the addition of SNJ1 were used as negative controls. Total genomic DNA was extracted and digested with SacI for Southern blot analysis. The primers SNJ1-southern-F and SNJ1-southern-R were designed to synthesise a probe corresponding to the region from 9243 to 9732 nt of the SNJ1 genome (Supplementary Table 4 ). The DNA probe preparation, hybridisation and detection were performed using a Detection Starter Kit II (Roche) according to the manufacturer’s instructions.
10
In Situ Detection of Hepatic HBV_circ_1
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The HBV_circ_1 probe-A (Table 1 ) targeting the HBV_circ_1 B site was labeled with DIG-11-dUTP by digoxin, using a Dig high primer DNA labeling kit and detection starter kit II (Roche). Hepatic tissue paraffin sections of HCC patients were treated with dewaxing and digested with protease K. Next, coverslips were hybridized in hybridization buffer (Roche) at 37°C overnight, and signals were detected using an Enhanced Sensitive ISH detection kit I (Boster, Wuhan, China). Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were then observed under a Leica DM2000 microscope (Leica, Wetzlar, Germany).
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