Journal
GENETICS AND MOLECULAR RESEARCH
Volume 12, Issue 4, Pages 4526-4539Publisher
FUNPEC-EDITORA
DOI: 10.4238/2013.October.15.1
Keywords
Genomic DNA extraction; Liquid nitrogen-free protocol; High-throughput protocol; Phenol-chloroform-free protocol; High-quality protocol; Real-time PCR
Funding
- National Key Technology R&D Program of China [2012BAD07B01]
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The isolation of high-quality genomic DNA (gDNA) is a crucial technique in plant molecular biology. The quality of gDNA determines the reliability of real-time polymerase chain reaction (PCR) analysis. In this paper, we reported a high-quality gDNA extraction protocol optimized for real-time PCR in a variety of plant species. Performed in a 96-well block, our protocol provides high throughput. Without the need for phenol-chloroform and liquid nitrogen or dry ice, our protocol is safer and more cost-efficient than traditional DNA extraction methods. The method takes 10 mg leaf tissue to yield 5-10 mu g high-quality gDNA. Spectral measurement and electrophoresis were used to demonstrate gDNA purity. The extracted DNA was qualified in a restriction enzyme digestion assay and conventional PCR. The real-time PCR amplification was sufficiently sensitive to detect gDNA at very low concentrations (3 pg/mu L). The standard curve of gDNA dilutions from our phenol-chloroform-free protocol showed better linearity (R-2 = 0.9967) than the phenol-chloroform protocol (R-2 = 0.9876). The results indicate that the gDNA was of high quality and fit for real-time PCR. This safe, high-throughput plant gDNA extraction protocol could be used to isolate high-quality gDNA for real-time PCR and other downstream molecular applications.
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