The UN resisted appointing Warren until the "last minute", with final approval only given on 1 March.
The general PCR format was: 94°C, 3 minutes; followed by cycles of 94°C for 30 seconds, gene-specific annealing temperature for 1 minute and 72°C for 1 minute; with final elongation at 72°C, 5 minutes.
First, we evaluated the effects on sperm motility of the incubation time (5 to 60 minutes) with different final concentrations (5%to20%0%) of glycerol, N-N-dimethylacetamide, dimethylsulfoxide (DMSO), ethylene glycol, propylene glycol, and methanol.
The cycling conditions were a 3-minute denaturation step at 94°C followed by 35 cycles at 92°C for 30 sec, 55°C for 30 sec and 68°C for 2.5 minutes with a final extension step at 68°C for 5 minutes.
Reverse transcription was performed at 25°C for 10 minutes, then 42°C for 60 minutes with a final incubation at 75°C for 15 minutes using gene-specific RT primer cocktail listed in the supplementary material (Table S1), a 5' adaptor (5'-GAC CAC GCG TAT CGG GCA CCA CGT ATG CTA TCG ATC GTG AGA TGG G-3') and PowerScript reverse transcriptase (Clontech, DB, USA).
The PCR was performed using Pfu UltraII Fusion HS DNA Polymerase (Stratagene, 600672) under the following conditions; 95°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 60°C for 30 seconds and 68°C for 10 minutes, with a final extension at 68°C for 10 minutes.
The reaction profile consisted of the following: 72°C for 5 minutes, followed by 25 cycles of 94°C for 1 minute, 55°C for 1.5 minutes and 72°C for 2 minutes, with a final extension step of 72°C for 10 minutes.
The thermocycler profile for the long PCRs consisted of one minute at 98°C, followed by 30 cycles of 98°C for 10 seconds and 68°C for 15 minutes, with a final extension of 10 minutes at 72°C.
The solvent system used 0.1% formic acid as solvent A and 100% methanol as solvent B, with a gradient elution run, beginning with 30% B for 0.5 minutes, ramping to 95% B over 1.5 minutes, holding at 95% B for 1 minute, and returning to 30% B in 0.25 minutes, with a final 30% B equilibration step for 2 minutes.
The normalized cDNA was then amplified from 1 μl of DSN-treated cDNA by PCR reactions (11 cycles) involving: 95°C for 1 minute, followed by 12 cycles with 95°C for 15 seconds, 64°C for 20 seconds and 72°C for 3 minutes, with a final extension of 72°C for five minutes and clean up with a QIAquick Mini Elute PCR column (Qiagen, Mississauga, ON, Canada).
