VIII. Tecnical Note
58. Silica gel mediated storage of rice leaf tissue and DNA
G.YANG, J. YANG, W. LI, C. QIN and Z. LUO
Dept. of Life Science, Hubei University, Wuhan, 430062 P.R. China
Development and utilization of molecular markers has advanced rice genetics. For genetic investigations DNA must be isolated from desirable amount of tissues depending on extraction scale. Traditional approaches use liquid nitrogen or dry ice for tissue collection and transportation. Fresh tissues are stored usually in -70°C and freeze dried for grinding. For extracting DNA from fresh tissues, liquid nitrogen is needed for homogenization (Zhang et al. 1994). In addition, DNA is not purified enough through CsCl ultracentrifugation or phenol extraction when handling large amount of samples. As a result, DNA cannot be stored for long term due to danger of its degradation. In this note, a reliable and convenient method is reported for storage of rice leaf tissue and DNA using silica gel without need of facilities mentioned above. It is extremely suitable for leaf tissue collection for mini-preparation of DNA. It also has unique advantage for sampling in field.
For normal scale DNA isolation, a 250 ml wide-mouth bottle with air-tight screw cap (for example, recycled food package bottles) containing about 50 gram silica gel is prepared. About 5 gram fresh leaf tissues are cut into small pieces and sealed in the bottle for about 24 hours. If gel becomes red, the tissues can be transferred into a fresh bottle. Water soaked silica gel can be regenerated by heating at 100°C for three hours or in microwave oven for 10 minutes. Leaf tissues dry thoroughly and can be stored in the bottles. Dried tissues are ground into powder with hand or grinder if available. The powder is transferred into 50 ml centrifuge tube for immediate DNA extraction or short term storage at room temperature.
CTAB protocol is used for DNA extraction (Saghai Maroof et al. 1984). In brief, tissue powder is stirred into emulsion with a glass rod after adding 10 ml extraction buffer prewarmed to 65°C (1% CTAB, 0.7 M NaCI, 50 mM Tris-HCI pH 8.0, 10 mM EDTA pH 8.0) and incubated at 65°C for one hour. Ten ml chloroform with isoamyl alcohol (24/ 1, v/v) is added and the mixture is shaken into emulsion again. The mixture is centrifuged at the highest speed available (for example, 7000 rpm in Hettich Universal 16R) for 10 minutes at 25°C. Supernatant is mixed with 10μl RNase (10 mg/ml) in a fresh tube and laid on bench for 10 minutes. DNA is precipitated with gentle shaking after adding 0.6 volume isopropanol. DNA is hooked out and transferred into 70% ethanol for washing. After brief drying on Whatman 3mm paper, DNA is dissolved in TE buffer for use.
For long term storage, DNA pellet can be wrapped into paper bag after brief drying and sealed in silica gel containing bottle at room temperature.
For mini preparation, one centimeter leaf segment of rice is dried and stored in a vial containing desirable amount of silica gel. Tissue is homogenized into emulsion in a small mortar (normal glass or porcelain mortar with pestle, top diameter 5 cm) with 500μl extraction buffer and transferred into a 1.5 ml centrifuge tube. Remaining steps are the same as that for normal scale DNA extraction except that buffers and solvents are reduced proportionally. DNA is precipitated through centrifugation and dissolved in 200 TE buffer. We use 2μl for a PCR reaction in RAPD and SSR analyses.
The quality of DNA isolated has been checked for molecular weight and uniformity using lambda DNA as reference. We found that DNA isolated from stored leaf tissues is uniform in size and has similar molecular weight as lambda. DNA can be used directly for restriction enzyme digestion and PCR reaction without further purification.
We gratefully acknowledge the financial support
from Wuhan Science and Technology Committee as one project of The Chenguang
Saghai Maroof, M.A., K.M. Solima, R.A. Jorgenson and R.W. Allard, 1984. Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. Proc. Natl. Acad. Sci. USA 81: 8014-8018.
Zhang, Q., Y.J. Gao, S.H. Yang, R.A. Ragab, M.A. Saghai Maroof and Z.B. Li, 1994. A diallel analysis of heterosis in elite hybrid rice based on RFLPs and microsatellite. Theor Appl Genet 89: 185-192