Genomic DNA extraction protocols from a Moroccan medicinal and aromatic plant Artemisia herba-alba Asso for RAPD-PCR studies

Summary of the parameters of RAPD-PCR tested and the optimum conditions selected. PCR parameter Range tested Optimum conditions DNA concentrations (ng) 5-10-20-30-40-50-70-100-200 5 ng Magnesium chloride (mM) 1.5-2.5-3-3.5-4-4.5-5 1.5 mM Primer concentration (µM) 0.1-0.3-0.5-0.7 0.3 µM Taq polymerase (units) 0.2-0.4-0.6-0.8-1- 1.2-1.4-1.6-1.8-2 0.4 unit dNTPs concentration (mM) 0.05-0.1-0.15-0.2-0.25 0.1mM Temperatures of annealing (°C) 33, 37 and 40 37°C Figure M 5 10 20 30 40 50 70 100 200 Figure 2. RAPD-PCR profile of nine DNA concentrations ranging from 5 to 200 ng obtained by the Operon primer OPA-05 in 1.8% agarose gel. M: 10 kb DNA ladder. M N 1.5 2 2.5 3 3.5 4 4.5 5 Figure 3. Optimization of RAPD-PCR parameters with eight MgCl 2 concentrations using the Operon primer OPA-05 and 5 ng of A. herba-alba genomic DNA. M: 10 kb DNA ladder. N: PCR negative control (no template DNA). optimization of different parameters for PCR (Weeden et al., 1992; Staub et al., 1996). There are difficult to get plant DNA free from contaminating proteins and polysaccharides. These compounds can link covalently to DNA which becomes useless in enzymatic based reactions. It is for that reason that our first efforts concerned the development of an extraction protocol capable of giving a DNA which can be used as support for the enzymatic reactions. To do it, two basic techniques were used. The technique of DNA extraction by the QIAGEN Kit, it is effective, simple and fast. The best adapted to the plant material of