title: pollen_July 17th 2012
design: 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 Pim Mix (45 mM Tris pH 8.3, 11 mM (NH4)2SO4, 4.5 mM MgCl2, 6.7 mM ß-mercaptoethanol, 0.5 mM dNTP’s, 0.11 µg/µl BSA fraction V, 4.4 µM EDTA) and 0.5 U of Phusion Polymerase, 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.
identifiers.namespace: Leiden University Medical Center
identifiers.id: pollen_July 17th 2012