Genomic DNA of transformants was extracted from 0.5 to 2.0 g of leaves using the Urea buffer. Fifteen micrograms of genomic DNA was digested with a restriction enzyme and separated by electrophoresis on a 1% agarose gel. The electrophoresed DNA was then blotted onto a nylon membrane (Pall Corp.). Digoxigenin-labeled DNA probes corresponding to 35S promoter were synthesized using a DIG DNA labeling kit (Roche). The DIG DNA probe was hybridized to the DNA at 45°C in DIG Easy Hyb solution. The membrane was washed twice with 2×SSC/0.1% SDS at room temperature and then twice with 0.5×SSC/0.1% SDS at 68°C. Visualization and detection were performed in the same way as that for Northern blot analysis.
Nylon membrane
Manufactured by Pall Corporation
Sourced in United States
The Nylon membrane is a type of lab equipment produced by Pall Corporation. It is a porous membrane made of nylon material. The core function of the Nylon membrane is to act as a filtration medium for various applications in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Dig dna labeling kit, by Roche (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
1200 series, by Agilent Technologies (1 mentions)
Heraeus megafuge 16r, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Dig labeling and detection system, by Roche (1 mentions)
Biotin 16 dutp, by Roche (1 mentions)
Hindiii, by Thermo Fisher Scientific (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Xhoi, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Expresshyb, by Takara Bio (1 mentions)
T4 polynucleotide kinase, by Takara Bio (1 mentions)
6 protocols using nylon membrane
1
Genomic DNA Extraction and Southern Blot Analysis
2
Spectrophotometric and HPLC Analysis
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Deionized (DI) water used in the process was generated from Purist® UV Ultrapure Water system (RephiLe Bioscience, Ltd., Boston, MA, USA), and a centrifuge (Heraeus™ Megafuge™ 16R, Thermo Fisher Scientific, Waltham, MA USA) was used to process the homogenized samples. For the spectrometric analysis, ion-exchange SPE cartridges (Oasis MAX 6 cc Vac Cartridge, 150 mg, 30 μm, Waters Corp., Milford, MA, USA) were first used to remove matrix interference from sample solutions, which were later analyzed on a spectrometer (NanoDrop™ 2000c, Thermo Fisher Scientific, Waltham, MA USA). For HPLC assay, each sample solution was filtered with a syringe filter (Nylon Membrane, 25 mm × 0.45 μm, Pall Corp., Port Washington, NY USA) and then analyzed on an HPLC system (Agilent Technologies 1200 Series, Santa Clara, CA USA) equipped with a C18 column (Zorbax® 5 μm SB-C18, 250 × 4.6 mm, Agilent Technologies, Santa Clara, CA USA).
3
Amino Acid Solution Preparation Protocol
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All AAs except for Gly were purchased from Applichem GmbH (Darmstadt, Germany). Gly was purchased from GERBU Biotechnik GmbH (Heidelberg, Germany). All AAs were dissolved in MNM to a final concentration of 3 mM (Tyr was first dissolved at 55 °C). MNM was prepared by adding 0.243 g K2HPO4, 0.234 g C6H12O6 and 0.491 g NaCl to 1,000 mL of deionized water (1.4 mM K2HPO4, 1.4 mM C6H12O6 and 8.4 mM NaCl), and then filtered by 0.22 μm syringe filters with nylon membrane (Pall Corp., USA).
4
Extraction and Analysis of Plant Nucleic Acids
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Genomic DNA was extracted from leaves using the High Pure GMO Sample Preparation Kit (Roche, Basel, Switzerland) according to the manufacturer's instructions. Total RNA was isolated from frozen leaf material (−80°C) using Trizol (Thermo Fisher Scientific, Carlsbad, CA, USA) and the RNeasy Kit (Qiagen, Hilden, Germany). Northern blots were carried out using the DIG labeling and detection system (Roche, Basel, Switzerland). Labeled probes comprising the entire CDS of OHP1 or OHP2 or approximately 300 bp of ELIP1 or ACT2 were generated by PCR, column-purified and diluted in high-SDS hybridization buffer (see Supplemental Table 1 for primer sequences). RNA separation, transfer to a nylon membrane (Pall Corp., Port Washington, NY, USA), hybridization and detection were performed as described by Woitsch and Römer (2003 (link)).
5
Viral DNA Extraction and Detection
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DNA was purified from virus-infected BSC-40 cells by phenol/chloroform extraction and ethanol precipitation, digested with XhoI or HindIII (Fermentas), and size fractionated on 0.7% agarose gels. The DNA was denatured in situ in a solution containing 0.5M NaOH and 1.5M NaCl, transferred to a nylon membrane (Pall Corporation), and fixed by UV cross-linking. A biotin-containing cro-gene probe was prepared using two primers (5’-TGATGGAACAACGCATAA-3’ and 5’-TTATGCTGTTGTTTTTTTGTTAC-3’), biotin-16-dUTP (Roche), template DNA, and the PCR. A second probe was purchased from IDT as a biotin-labeled oligonucleotide (5’-biotin-AAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAA-3’). It detects the synthetic early-late promoter driving mCherry gene expression. The probes were hybridized to the prepared membrane using ExpressHyb (Clontech), and detected using streptavidin conjugated to IRDye 800CW (Li-Cor) and an Odyssey Li-Cor scanner.
6
Detection of miRNA-124a in Mouse Tissues
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The PAGE Northern blot analysis was performed as described previously (2 (link)). Briefly, total RNAs from mouse tissues were isolated by Trizol (Invitrogen), and 20 μg of the total RNAs were denatured in 5 mM EDTA containing formamide at 80 °C for 5 min. RNAs were separated on 15% denaturing (7 M urea) polyacrylamide gels. RNAs were transferred to a nylon membrane (Pall Corporation Biodyne). LNA-modified anti–miR-124a (Exiqon, 20 pmol) was end-labeled with γ-32P-ATP (Muromachi Yakuhin) using T4 polynucleotide kinase (Takara). The nylon filters were hybridized with the labeled probe in salmon sperm–containing hybridization solution (120 mM sodium phosphate (pH 7.2), 250 mM sodium chloride, 7% SDS, and 50% formamide) at 43 °C overnight. The filters were exposed to X-ray film.
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