Genomic DNA from PSU was digested with the endonucleases AluI, HhaI, and MboI. Genomic DNA of the other studied species (CCR, PER, PRO, and RNO) was digested with AluI and MboI. The resulting fragments were separated in an agarose gel and blotted onto a Nylon membrane Hybond-N+ (Amersham, GE Healthcare). The membranes were then probed with the cloned PSUcentSat sequence, previously labeled by PCR with digoxigenin-11-dUTP (Roche Diagnostics). Hybridization was performed at 42 °C in hybridization solution (Roche Diagnostics). The positive signals were visualized using chemiluminiscent CDP-Star system (Roche Diagnostics). Selection of REs was performed using the CLC Sequence Viewer software (version 6.2, http://www.clcbio.com/index.php?id=28 , last accessed October 27, 2014).
Hybridization solution
Manufactured by Roche
Sourced in Switzerland, Germany
Hybridization solution is a laboratory reagent used to facilitate the binding of nucleic acid molecules, such as DNA or RNA, to their complementary sequences during hybridization experiments. It is a critical component in various molecular biology techniques, including Northern blotting, Southern blotting, and in situ hybridization.
Automatically generated - may contain errors
Lab products found in correlation
Cdp star system, by Roche (1 mentions)
Hybond n nylon membrane, by Cytiva (1 mentions)
Digoxigenin 11 dutp, by Roche (1 mentions)
Nylon membrane, by Roche (1 mentions)
S1 nuclease, by New England Biolabs (1 mentions)
5 bromo 4 chloro 3 indolyl phosphate and nitro blue tetrazolium bcip nbt, by Promega (1 mentions)
Ap labeled anti dig antibody, by Roche (1 mentions)
Te2000 u microscope, by Nikon (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Hybond n membrane, by Cytiva (1 mentions)
Dig northern starter kit, by Roche (1 mentions)
5 protocols using hybridization solution
1
Comparative Genomic DNA Analysis
2
Determining plasmid-borne bla_CMY-2 gene
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To determine whether blaCMY-2 was located in the chromosome or on a plasmid, S1 nuclease (New England BioLabs) and BlnI (Takara Bio, Otsu, Japan) digestions were prepared from the total DNA of the four isolates selected for ISEcp1 analysis (harboring blaCMY-2) (S. Infantis isolates 1993, 2127, and 2150, and S. Manhattan isolate 2179), as well as two susceptible isolates (S. Infantis isolate 1737 and S. Manhattan isolates 2129) for use as blaCMY-2-negative controls. Total DNA was treated with 2 U/ml of S1 nuclease (incubated at 37°C for 45 min) or BlnI (incubated at 37°C for 16 h), followed by PFGE separation [30 (link)]. Separated fingerprints were transferred to positively-charged Nylon membranes (Roche Applied Science, Penzberg, Germany) and hybridized with PCR-generated blaCMY-2 digoxigenin-labeled probes (Roche Diagnostics, Basel, Switzerland) using hybridization solution (Roche Diagnostics) according to the manufacturer’s instructions and a previous report [32 (link)].
3
Placental miRNA-21 Expression in GDM
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Human placentas from 137 GDM patients and 158 normal pregnant women were separately analyzed using two paraffin tissue microarrays according to the match between the case- and the control group. Paraffin sections were dewaxed and hydrated, denatured with 100μg/mL proteinase K (TransNGS, Beijing, China), and fixed in 4% paraformaldehyde. After pre-hybridization of the sections with the hybridization solution (Roche, Mannheim, Germany) at 40°C for 1 h, the digoxigenin (DIG)-labeled LNA-miR-21 probe was incubated at 40°C overnight. It was then washed with physiological saline drop sodium citrate (SSC) and blocked with blocking buffer containing 5% bovine serum albumin (BSA). The sections were incubated with alkaline phosphatase (AP)-labeled anti-DIG-antibody (Roche, Mannheim, Germany, 1:250) overnight at 4°C, and developed with 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium (BCIP-NBT; Promega, Madison, WI, USA). Samples were viewed using a Nikon TE 2000-U microscope (NIKON, Tokyo, Japan).
4
Southern Blot Hybridization Protocol
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Genomic DNA was isolated by using the DNeasy Blood and Tissue kit (Qiagen). For Southern hybridization, 10 µg of genomic DNA was used, subjected to electrophoresis in agarose gels and transferred to a positively charged nylon membrane (Roche, Basel CH) according to manufacturer instructions. The hybridized DNA was detected according to the instructions of the manufacturer (Roche) by using the digoxigenin (DIG) non-radioactive nucleic acid labeling and detection system. DNA was labeled with DIG-dUTP by using Klenow fragment, and the DIG-labeled DNAs were used as probes for experiments after they were made single stranded by boiling for 10 min, followed by chilling in ice. Blots were incubated with the labeled probes for 16 h at 65°C in hybridization solution (Roche). The membranes were prewashed twice at room temperature with 2 × SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) containing 1% (wt/vol) SDS for 5 min, and this was followed by two stringency washes with 0.1× SSC–0.1% SDS for 15 min at 65°C. Color detection of the hybridized probes was carried out by using the instructions of the manufacturer (Roche) and NBT/BCIP as the detection reagent.
5
Northern blot analysis of plant genes
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Total RNA was fractionated on a 1.5% denaturing agarose gel containing formaldehyde and transferred onto Hybond N+ membranes (Amersham, UK). Hybridizations were carried out at 68 °C overnight in hybridization solution (Roche). PCR-amplified fragments of orf147, orf133 and nad7 gene fragments were used as a probe after labeling with the nonradioactive DIG Northern Starter Kit (Roche, Germany). Labeling, hybridization and detection were performed according to the manufacturer's instructions.
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