Platinum direct pcr universal master mix kit

Multiple taxonomic gene regions for all studied lichens were selected and amplified based on the latest phylogenetic-based studies available for each lichen genus as outlined in detail below. All PCR reactions were carried out using the Platinum Direct PCR Universal Master Mix - Kit (Thermo Fisher Scientific Inc) in a Mini Amp Plus Thermal Cycler (Applied Biosystems, Thermo Fisher Scientific, Waltham, USA).
All PCR products were checked by gel electrophoresis using 1% E-gel EX (Invitrogen, Thermo Fisher, Waltham, USA) in an E-Gel Power Snap instrument (Invitrogen, Thermo Fisher, Waltham, USA).
Successful PCR products were purified using the NucleoSpin Gel and PCR Clean-up Kit (Marchery Nagel, New England, Canada) according to the manufacturer’s standard protocol. Subsequently, they were sent for Sanger sequencing carried out by Azenta (Göttingen, Germany) with the primers that were for the amplification of the respective gene regions. All generated DNA sequences were deposited at the National Center for Biotechnology Information (NCBI) GenBank.

Biomass from three replicates per species were picked with tweezers under a binocular stereoscope and subsequently washed in sterile ddH₂O. Small proportions of these (equivalent to the size of three sand grains) were finally picked off the cleaned lichen fragments and transferred to 200 µl PCR tubes filled with lysis buffer of the Platinum Direct PCR Universal Master Mix - Kit (Thermo Fisher Scientific Inc). Lysis was carried out according to standard protocol provided by the manufacturer and acted as template for subsequent PCRs.