Reaction fractions were treated by protease K (50 µg/ml) for one hour at 55°C and were then submitted to phenol/chloroform/isoamylalcohol (25/25/1, v/v/v) extraction.
First we evaluated biodiesel reaction fractions from MBM spiked with 5% uninfected brain (MBM c) for potential toxic effects in our animal bioassay.
Crude reaction fractions of biodiesel and glycerol were diluted with 4x sample loading buffer (SLB, Invitrogen Corp., Carlsbad, California, United States), heated to 70°C for 10 min and directly loaded onto a precast 4 12% gradient gel (Invitrogen Corp).
Products were detected only after proteinase K treatment of the reaction fractions, consistent with the fact that tyrosine recombinases are known to bind very tightly with DNA which is released from complexes after proteinase K digestion.
Absorbance at 475 nm for both control and experiment reaction fractions were recorded for each 5 min interval for 20 min.
For the above reaction, fractions eluted at 19 22 (sample A), 26 30 (sample B), and 31 34 min (sample C) were combined and concentrated to dryness using a speedvac.
The process design consisted of several units (reaction, solid fraction washing, products recovery and liquid fraction processing).
The simulation design and treatment sequences (reaction, solid fraction washing, products recovery and liquid fraction processing) are similar for both processes.
The second reaction mix fraction (F2) consisted of 4.4 µl of 2X reaction mix, 200 nM of forward primer (F56_1 3 or gen2FSN), 1.5 µl of 50 mM MgSO4, 0.1 µl of ROX (25 µM), and 200 nM of each probe (against either genotypes 1a, 1b, and 3a; or against genotypes 2a, 2b, and 2c).
After 1 h of binding reaction, the fraction of protein in the supernatant and the pellet was estimated by 12% SDS-PAGE analysis.
