10,000×g for 1 minute and the supernatant was discarded. 9- Column washing: Wash buffer (750 was gently washed through the column and μl) centrifuged at 10,000×g for 1 minute and the flow through discarded. The column was centrifuged at 10,000×g for 1 minute to removed residual ethanol. 10- DNA elution: a clean microcentrifuge tube was used to collect DNA by adding 100 of preheated elution buffer directly into the column membrane and standing for 2 μl minutes at room temperature and then centrifuging at 10,000×g for 1 minute to elute DNA. DNA was stored at -20°C. 3-2-3 the Polymerase Chain Reaction (PCR) The Polymerase Chain Reaction was used during this work for amplifying specific 16SrRNA genes PCR was prepared as follows: 3-2-3-1Reaction mixer of PCR product 3-2-3-2 Quantity Types Distilled water 38μl, 5.0 10x buffer, 2.5 50mM Mgcl2, 0.5 of Forward Primer ( μl μl μl 16S rRNA unv. 10pp mol.L-1) (Weisburg et 1991) 0.5 of Reverse Primer (16S al., μl rRNA unv. 10 pp mol.L-1) (Weisburg 1991), 1.0 dNTPs, 2.0 Genomic DNA et al., μl μl and 0.5 Bioline Taq. PCR was running on the following cycle (Named 16s Standard) μl 3-2-3-3 PCR cycle Initial denature 94°C for 3 min to separate the double strand of DNA to single strand RNA.Denature 94°C for 1 min. Annealing 60°C for 1 min to allow primers to match
10,000×g for 1 minute and the supernatant was discarded. 9- Column washing: Wash buffer (750 μl) was gently washed through the column and centrifuged at 10,000×g for 1 minute and the flow through discarded. The column was centrifuged at 10,000×g for 1 minute to removed residual ethanol. 10- DNA elution: a clean microcentrifuge tube was used to collect DNA by adding 100 μl of preheated elution buffer directly into the column membrane and standing for 2 minutes at room temperature and then centrifuging at 10,000×g for 1 minute to elute DNA. DNA was stored at -20°C. 3-2-3 the Polymerase Chain Reaction (PCR) The Polymerase Chain Reaction was used during this work for amplifying specific 16SrRNA genes PCR was prepared as follows: 3-2-3-1Reaction mixer of PCR product 3-2-3-2 Quantity Types Distilled water 38μl, 5.0 μl 10x buffer, 2.5 μl 50mM Mgcl2, 0.5 μl of Forward Primer ( 16S rRNA unv. 10pp mol.L-1) (Weisburg et al., 1991) 0.5 μl of Reverse Primer (16S rRNA unv. 10 pp mol.L-1) (Weisburg et al., 1991), 1.0 μl dNTPs, 2.0 μl Genomic DNA and 0.5 μl Bioline Taq. PCR was running on the following cycle (Named 16s Standard) 3-2-3-3 PCR cycle Initial denature 94°C for 3 min to separate the double strand of DNA to single strand RNA.Denature 94°C for 1 min. Annealing 60°C for 1 min to allow primers to match