A high-throughput, high-quality plant genomic DNA extraction protocol.
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 µg 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/µL). 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.
Key Laboratory of Ion Beam Bioengineering, Institute of Technical Biology and Agriculture Engineering, Chinese Academy of Sciences, Hefei, China.
H
H
Li
J
J
Li
X H
XH
Cong
Y B
YB
Duan
L
L
Li
P C
PC
Wei
X Z
XZ
Lu
J B
JB
Yang
D029688:Brassica napus; D018744:DNA, Plant / Q000737:chemistry / Q000302:isolation & purification*; D018745:Genome, Plant; D012275:Oryza / Q000737:chemistry; D018515:Plant Leaves / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards; D012015:Reference Standards; D015183:Restriction Mapping; D014908:Triticum / Q000737:chemistry; D003313:Zea mays / Q000737:chemistry
D018744:DNA, Plant