Embrapa Maize And Sorghum Lab Report
Experimental sites and Isolation of endophytic bacteria from plant tissues of maize
The work was carried out in the year of 2013, at Embrapa Maize and Sorghum Research Center of the Embrapa (Brazilian Agricultural Research Corporation), located in Sete Lagoas City (19° 27′ 57″ S; 44° 14′ 49″ W), Minas Gerais State, Brazil. The climate is characterized by highland tropical climate (hot and warm summers), and dry winters, savannah type, according to the Köppen-Geiger classification (Peel et al., 2007), with average annual rainfall of 1.403 inches and the average annual temperature between 22 to 27 oC.
Leaves samples, stem, and roots of maize plant collected during the flowering stage were thoroughly washed under running water and sterilized by …show more content…
The PCR reaction mixture consisted of 5 μL DNA (10 ng/μL), 5 μL of 10X PCR buffer, 2.5 μL MgCl2 (50 mM), 2.0 μL dNTP's (2.5 mM each), 4.0 μL each primer (5 μM), 0.3 μL Taq DNA Polymerase (5 UμL-1, Invitrogen), 0.5 μL formamide, and 26.7 μL Milli-Q water in a total volume of 50 μL. The enzymatic amplification wasperformed using an automated Veriti Thermal Cycler (Applied Biosystems). Prior to PCR cycling, the reactions were incubated at 94 °C for 1 minute to fully denaturate the DNA. Afterwards, the DNA amplification was carried out by 30 cycles of 1 min at 94 °C, 1 min at 55 °C (annealing), and 2 min at 72°C (extension). Finally, the reactions were incubated for 10 minutes at 72°C. The Amplified DNAs were analyzed by horizontal gel electrophoresis at 100 V in 1.5% agarose gel (wt/v) in 1X TAE buffer (0.04M Tris-Acetate, 0.001M EDTA, pH 8.0), stained with 2.5X solution of GelRed™ Nucleic Acid Gel Stain (250μL GelRed™ to 1 liter of distilled water), and gels were visualized under UV light and photographed. Afterwards the PCR amplified products were cut out from the gels and purified using the QIAquick Gel Extraction kit (Qiagen). Sequencing reactions were performed with the BigDye Terminator v3.1 Cycle Sequencing Kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA), and DNA sequencing was resolved on an Applied Biosystems automatic sequencer ABI-3100 (Applied Biosystems). Sequences editing and contig sequences were generated with ClustalW (Thompson et al., 1994) and CAP3 Program (Huang & Madan, 1999) and similarity was evaluated by the BlastN program (Altschul et al., 1997) run against all available DNA sequences deposited in the GenBank database (http://www.ncbi.nlm.nih.gov). Nucleotide sequences of bacteria and fungi were deposited in EMBL/GenBank/DDBJ nucleotide
The work was carried out in the year of 2013, at Embrapa Maize and Sorghum Research Center of the Embrapa (Brazilian Agricultural Research Corporation), located in Sete Lagoas City (19° 27′ 57″ S; 44° 14′ 49″ W), Minas Gerais State, Brazil. The climate is characterized by highland tropical climate (hot and warm summers), and dry winters, savannah type, according to the Köppen-Geiger classification (Peel et al., 2007), with average annual rainfall of 1.403 inches and the average annual temperature between 22 to 27 oC.
Leaves samples, stem, and roots of maize plant collected during the flowering stage were thoroughly washed under running water and sterilized by …show more content…
The PCR reaction mixture consisted of 5 μL DNA (10 ng/μL), 5 μL of 10X PCR buffer, 2.5 μL MgCl2 (50 mM), 2.0 μL dNTP's (2.5 mM each), 4.0 μL each primer (5 μM), 0.3 μL Taq DNA Polymerase (5 UμL-1, Invitrogen), 0.5 μL formamide, and 26.7 μL Milli-Q water in a total volume of 50 μL. The enzymatic amplification wasperformed using an automated Veriti Thermal Cycler (Applied Biosystems). Prior to PCR cycling, the reactions were incubated at 94 °C for 1 minute to fully denaturate the DNA. Afterwards, the DNA amplification was carried out by 30 cycles of 1 min at 94 °C, 1 min at 55 °C (annealing), and 2 min at 72°C (extension). Finally, the reactions were incubated for 10 minutes at 72°C. The Amplified DNAs were analyzed by horizontal gel electrophoresis at 100 V in 1.5% agarose gel (wt/v) in 1X TAE buffer (0.04M Tris-Acetate, 0.001M EDTA, pH 8.0), stained with 2.5X solution of GelRed™ Nucleic Acid Gel Stain (250μL GelRed™ to 1 liter of distilled water), and gels were visualized under UV light and photographed. Afterwards the PCR amplified products were cut out from the gels and purified using the QIAquick Gel Extraction kit (Qiagen). Sequencing reactions were performed with the BigDye Terminator v3.1 Cycle Sequencing Kit according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA), and DNA sequencing was resolved on an Applied Biosystems automatic sequencer ABI-3100 (Applied Biosystems). Sequences editing and contig sequences were generated with ClustalW (Thompson et al., 1994) and CAP3 Program (Huang & Madan, 1999) and similarity was evaluated by the BlastN program (Altschul et al., 1997) run against all available DNA sequences deposited in the GenBank database (http://www.ncbi.nlm.nih.gov). Nucleotide sequences of bacteria and fungi were deposited in EMBL/GenBank/DDBJ nucleotide