Assays were conducted in triplicate on an ABI 7500 Real Time instrument using the following conditions; 50° for 2 minutes, 95° for 10 minutes, and then 95° for 15 seconds and 60° for 1 minute repeated 40 times.
Thermal cycling conditions were as follows: Denature at 95°C×30 seconds, anneal at 54°C×30 seconds and extend at 72°C×1 minute (repeat for 25 cycles), final extension at 72°C×8 minutes.
Briefly, nub-RD cDNA was amplified from the expression plasmid pAct-Pdm1[ 35] using Pfu DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and the following conditions: 95°C for 3 minutes; 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 4 minutes, repeated 30 cycles; 72°C for 5 minutes.
If it doesn't fit, or it doesn't look right, put it back into the hot water for a minute, repeat the shaping steps until it turns out the way you want it.
Each exercise lasted 1 minute and was repeated for three times; a 30-second recovery pause was set between each series.
Each loop of song was preceded by a silent gap of 3 minutes and then repeated for two more times to complete a song stimulus of 12 minutes (3 loops of 2 minutes song - 3 minutes silence).
All told a three- or four-minute procedure -- repeated for several passengers -- that could slow check-in times on Southwest's normally busy schedule, which includes flights to some destinations every half-hour.
Subjects were removed from the restraint device following the 30 minute sample and placed in a transport box until the 60 minute sample, and this was repeated for the 120 minute sample.
In cell sorting experiments, the epithelium was discarded and the deepithelialized tissue was then rinsed and incubated with RPMI 1640 medium-5% FCS-HEPES-antibiotic/antimycotic at 37°C for 10 minutes, repeated 1 time.
The immobilised metal affinity capture (IMAC30) chip was chosen for the actual screening and was pre-treated with nickel before analysis: 5 ul 100 mM NiSO4 were added to each spot and left on shaker (250 rpm) for 5 minutes (repeated twice).
