Wizard sv 96 pcr clean up system

To determine the ITS sequence of representative isolates from the 32 different soil samples, we randomly selected 245 isolates from the 1556 isolates to include both nosZ+/− isolates based on the incidence of the nosZ genotype (Fig. S1, Table S3). The total DNA lysate was used as the template DNA in a 50-μL reaction mixture for PCR using ExTaq DNA polymerase. In the ITS amplification, the ITS primer set and PCR cycle conditions were the same as those described previously (23 (link)). Amplified DNA fragments were purified using the Wizard SV 96 PCR Clean-Up System and Vac-Man 96 Vacuum Manifold (Promega, Madison, WI, USA). The sequencing of amplified DNA fragments was performed by the Dragon Genomics Center at TAKARA BIO INC. (Otsu, Japan). The BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) was used for the sequencing reaction, and DNA sequencing was then carried out on an ABI 3730xl DNA Analyzer (Applied Biosystems).

Sorted yeast populations were plated on SD -Leu and 8–32 colonies per plate were picked into 2 mL microplates either by hand or using a QPIX 420 (Molecular Devices) automatic colony picker. Cultures were grown at 1000 rpm at 3 mm orbit at 30°C overnight. Cells (20 μL) were transferred to a fresh microplate and washed with 1 mL TE buffer (10 mM Tris, 1 mM EDTA). Cells were incubated with 20 μL zymolyase solution (5 mg/mL zymolyase, 100T in TE) at 37°C for 1 hour. Cells (5 μL) were then used in colony PCR to amplify the paired heavy and light chains. Amplicons were column purified with the Wizard SV 96 PCR Clean-Up System (Promega, cat. no. A9342) and yields were quantified with a Nanodrop spectrophotometer or the Quant-it Broad-Range dsDNA kit (Invitrogen, cat. no. Q33130). Approximately 10 ng (2.5–5 μL) of purified PCR products were then subjected to Sanger sequencing.

Maternal DNA was extracted with a Qiagen kit according to manufacturer recommendations (Qiagen, Valencia, CA, US). At least 1 ng/μl DNA was amplified with a forward primer (FWD 5′-GTCGGGTGCTGACACATCTG-3′) and a reverse primer extended by a universal primer M13 (REV 5′-AGCGGATAACAATTTCACACAGGA | CTTGCCGGCYRTSGCACTC-3′) [26 (link)]. The additional M13 sequence extended the amplicon size in order to enhance sequence quality. The amplification was performed with AccuPrime Taq DNA Polymerase, High Fidelity (Fisher Scientific, Florence, KY, US) and the buffer II (delivered with the enzyme). PCR product purification was performed with the Wizard SV 96 PCR Clean-Up System (Promega, Madison, WI, US). For the sequencing, the amplification forward (FWD) and/or a second forward (FWD2 5′-AGGTCAGCCTGACCTGCCTG-3′) primer [26 (link)] was used for sequence accuracy and led to 805- and 277-nucleotide-long sequences, respectively (from bp 1,382 or 1,910 to bp 2,186; accession number NC_000014.9). In case of a nonconclusive sequencing, a third reverse primer was used (M13-REV 5′-AGCGGATAACAATTTCACACAGGA-3′). The sequencing was performed by Eurofins Genomics (Louisville, KY, US).