pollen_May 26th 2012
TrnL primers with Ion Torrent tails were used for the PCR amplification. The forward primer was a combination of the Ion Torrent A adapter with trnL-c (Taberlet et al. 1991, 2007), and the reverse primer the Ion Torrent P1 adapter along with trnL-h ((Taberlet et al. 2007). The length of the target sequence varied between species, but averaged about 200 bp.  PCR was performed in a total volume of 25 μl containing 1x Phusion High Fidelity PCR Master Mix (New England Biolabs), 250 nM of each primer and 1 μl of DNA. PCR conditions were 30 seconds at 98ºC, 25 cycles of 10 seconds at 98ºC, 30 seconds at 61ºC, and 30 seconds at 72ºC, followed by 5 minutes at 72ºC.
Leiden University Medical Center
pollen_May 26th 2012