Favorprep pcr purification kit

Non-starter MJ samples were collected at day 0, 14, and 28. Total DNA was extracted by using a EZ-10 Spin Column Soil DNA Miniprep Kit (Bio Basic Inc., Canada). 16S rRNA genes were amplified from the extracted DNA samples by using universal primer pair, 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-GGY TACCTTACGACTT-3'). PCR was done under the following conditions: denaturation at 94 o C for 5 min, 40 cycles of 30 sec at 94 o C, 2 min at 57 o C, and 2 min at 72 o C, and a final extension at 72 o C for 7 min. Amplified fragments were purified by using a FavorPrep PCR Purification Kit (Favorgen, Taiwan) and ligated with pGEM-T easy vector (Promega, USA). Escherichia coli DH5α competent cells (Enzynomics, Korea) were transformed with the ligation mixture, and transformants were selected on LB plates with ampicillin (100 μg/ml), isopropyl β-D-1-thiogalactopyranoside (500 μg/ml), and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (80 μg/ml). Thirty colonies were selected randomly from each MJ sample, and their inserts were sequenced. DNA sequencing was done at Cosmogenetech (Korea), and BLAST was used to find homologous sequences in the data library (http://www.ncbi.nlm. nih.gov/BLAST).