The RFLP analysis was performed on qPCR-ML products with EcoRI restriction enzyme (Roche Life Sciences, Indianapolis, USA), following the manufacturer’s protocol. The EcoRI digestion was performed at 37 °C for 2 h in a 20 μl mixture containing 7 U of enzyme and 50–100 ng of PCR products. The digestion mixtures were analysed on a 3% high-resolution MetaPhor (Cambrex, East Rutherford, USA) agarose gel, and the selected products were excised, purified with MinElute PCR purification kit (Qiagen) and sequenced as described above.
Ecori restriction enzyme
Manufactured by Roche
Sourced in United States
EcoRI is a type II restriction enzyme that recognizes and cleaves the palindromic DNA sequence 5'-GAATTC-3' and its reverse complement 5'-CTTAAG-3'. It is widely used in molecular biology research and applications.
Automatically generated - may contain errors
3 protocols using ecori restriction enzyme
1
RFLP Analysis of qPCR-ML Products
2
Identification of Ubi:CaPUB1 Transgenic Lines
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To identify independent lines of Ubi:CaPUB1 transgenic plants, a genomic Southern blot analysis was performed. Total genomic DNA was extracted from leaves using CTAB solution (2% CTAB, 2% PVP-40, 1.4 M NaCl, 100 mM Tris-HCl, pH 8.0, and 20 mM EDTA). Total DNAs from wild-type and transgenic plants were digested with EcoRI restriction enzyme (Roche, USA), precipitated, and separated by electrophoresis on 0.8% agarose gels. The gels were serially soaked in depurination, denaturation, and neutralization buffer. DNA on the gel was transferred to a nylon membrane (GE healthcare, Sweden) via the capillary transfer method. The DNA gel blot was hybridized with 32P-labeled hygromycin B phosphotransferase (HPT) probe under high-stringency hybridization and washing conditions (65°C). The autoradiography signals were visualized using the Bio-Imaging Analyzer (BAS2500; Fuji Film, Japan).
3
Plasmid DNA Purification and Linearization
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Plasmid DNA was purified through caesium chloride gradient centrifugation as reported in [14 (link),18 (link),21 (link)]. A stock solution of the plasmid 0.7 μM in milliQ water (Millipore Corp., Burlington, MA, USA) was stored at −20 °C. Linearized plasmid DNA (pEGFP-C1, Clontech, Takara Bio USA Inc., Mountain View, CA, USA) was obtained by cutting with EcoRI restriction enzyme (Roche, Monza, Italy), column purified (Genomed, Leesburg, FL, USA) and precipitated in alcohol. The pellet of the linearized plasmid DNA was dissolved in distilled water at a final concentration of 1 μg/μL, after washing with 70% of ethanol and air drying.
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