DNA sequence of negative control of PTFE membrane (DNA isolation)
As negative control of PTFE membranes, the DNA was isolated from clear PTFE membranes using DNeasy PowerWater Kit (Qiagen, Germany). The forward primer 5.8S_Fun 5-AACTTTYRRCAAYGGATCWCT-3 and reverse primer ITS4_Fun 5-AGCCTCCGCTTATTGATATGCTTAART-3 were used. The first PCR reaction mix contained nuclease-free water, 1 M of each of the appropriate primers, 0.02 U/l KAPA HiFi HotStart ReadyMix and 2 l of template in a total reaction volume of 15 l. Each sample was made in 8 replicates. The following thermocycling program was used: 5 min of denaturation at 95 C, followed by 30 cycles of 20 s at 98 C, 15 s at 50 C and 15 s at 72 C. Final extension was run at 72 C for 5 min. Next, the replicates were merged and concentrated using the commercial kit Genomic DNA Clean & Concentrator (ZYMO Research, Irvine, CA, USA). The second PCR reaction mix contained nuclease-free water, 1 M of each of the appropriate primers, 0.02 U/l KAPA HiFi HotStart ReadyMix and 1 l of template in a total reaction volume of 25 l. The following thermocycling program was used: 5 min of denaturation at 95 C, followed by 13 cycles of 20 s at 98 C, 15 s at 50 C and 15 s at 72 C. Final extension was run at 72 C for 5 min. Next, amplicons were sent to the Core Facility for Nucleic Acid at the University of Alaska Fairbanks for Illumina MiSeq platform sequencing.