The concentration of DNA in negative controls was measured at 260 nm using
a NanoDrop spectrophotometer. The PCR mixture (25 μL) Bafilomycin A1 datasheet was composed of 12.5 μL of 2 × Combi-PPP mix (Top-Bio Ltd, Prague, Czech Republic, contains hot start-Taq DNA polymerase, 5 mM MgCl2, buffer, deoxyribonucleotides and loader), 0.5 μL 10 μM forward primer, 0.5 μL 10 μM reverse primer, 0.5 μL DNA template and 11 μL water. Thermal programs for primer pairs used in this study are given in Table 1. Nested PCR directed to ITS region was performed using primer pair NSI1/NLB4 in the first amplification and either the pair Tu1sekvF/Tu2sekvR or the pair UncI/UncII in the second. Nested PCR directed to the β-tubulin gene was performed using primers Bt2a/Bt2b in Dasatinib mouse the first amplification and primers tubtubf/elytubr in the second. The annealing temperature originally recommended for
this primer pair is 63 °C, but with this temperature the PCR was not sufficiently sensitive for T. aestivum DNA and the annealing temperature was therefore decreased as indicated in Table 1. In addition, nested PCR was performed using the primers Bt2a/BTAEMB-R in the first amplification and BTAE-F/Bt2b in the second. The same thermal program as indicated for the primer pair BTAE-F/BTAEMB-R in Table 1 was used in both steps of amplification but the annealing temperature was set to 56 °C. The product of the first amplification was always diluted 1 : 100 before being used as a template in the second amplification. Templates were prepared by the Ribose-5-phosphate isomerase addition of small amounts of T. aestivum DNA (extracted from the sample S13, see Appendix S1) into complex nontarget DNA (negative control A). Resulting mixtures contained 2.5, 0.25, 0.025, 0.0025,
0.00025 or 0.000025 ng S13 DNA and 24.5 ng nontarget DNA in 1 μL water. These mixtures were used in nested PCR with primer pairs NSI1/NLB4 (first amplification) and Tu1sekvF/Tu2sekvR (second amplification) as indicated above with annealing at 59 °C. A 5-μL aliquot of the product of PCR amplified using the Tu1sekvF/Tu2sekvR primer pair was mixed with 9 μL water, 1 μL buffer R and 5 U of TaiI restriction endonuclease (New England Biolabs Inc., Ipswich, MA). The mixture was then incubated for 3 h at 65 °C and immediately separated on agarose gel. Soil and ectomycorrhizae samples were collected in the native habitat of T. aestivum, Chuchelský háj, near Velká Chuchle, Prague, Czech Republic. Plant cover was dominated by Carpinus betulus with addition of Fraxinus excelsior, Corylus avellana and Tilia cordata seedlings. Twelve 200 g soil samples were collected on an L-shaped terrain transect at 1 m equidistant points (Fig. 1) from the depth of 0–10 cm (A-horizon, rendzina on silurian lime). Ectomycorrhizae were separated manually from the soil sample.