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DNA Yields and Optimization of RAPD Patterns Using Spruce Embryogenic Lines, Seedlings, and Needles

Nkongolo, K.K., Klimaszewska, K., Gratton, W.S.
Plant molecular biology reporter 1998 v.16 no.3 pp. 284
DNA, Picea, conifers, genetic analysis, random amplified polymorphic DNA technique, seedlings, tissue culture
Genetic analysis of plants relies on high yields of pure DNA samples. In conifers, such DNA samples are usually derived from tissue cultures. However, it is labourious to use embryogenic cultures for projects that require large sample numbers because the starting material has to be initiated from the seed in vitro. As an alternative, we have used seedlings as source material for DNA extraction and have obtained yields and purities comparable to embryogenic cultures. RAPD profiles obtained with DNA from embryogenic cultures and seedlings were similar and highly reproducible. Efficient and consistent amplification was achieved using primer concentrations ranging from 1.6 and 6.4 μM with 0.1 to 1.0 μg of template DNA. The intensity and resolution of faint bands resulting from non-optimized conditions were easily improved through further rounds of synthesis in a fresh PCR with amplified DNA diluted 500-fold.