Qiaquick pcr purfication kit

To confirm the isolate was O. unilateralis
s.l., small subunit (SSU) was
amplified, sequenced, and blasted using the Megablast algorithm against the entire
Fungi (taxid:4751) nucleotide collection (nr/nt) (blastn, NCBI). Genomic DNA
extraction was performed on flash frozen fungal mycelium mechanically disrupted
inside a frozen 2 mL eppendorf with two 5/32 inch metal balls (Wheels
Manufacturing Inc.) using a TissueLyser II (Qiagen) and a chilled adapter set
(Qiagen) at 24 freq/sec for 60 sec. DNA was taken up in 900 μL extraction buffer
(1% SDS, 155 mM 4-aminosalicyclic acid and 0.2 mL/mL 5× RNB (1.0 M Tris, 1.25 M
NaCl, 0.25 M EGTA, pH 8.5)), and 900 μL phenol:chloroform:isoamyl alcohol 25:24:1.
After centrifugation for 10 min. at 10.000 × g the water phase was taken up in 5
volumes PB buffer (Qiagen) and the DNA was purified using the columns, reagents
and protocol of the QIAquick PCR Purfication Kit (Qiagen). SSU was amplified with primers NS1 and NS4 [58 ] using Platinum Taq DNA polymerase
(Invitrogen), reaction conditions advised by the manufacturer of the enzyme, and
the cycler program described in [59 (link)].
The PCR product was sequenced by Macrogen (Maryland, USA) with the same primers
for initial amplification.

Total genomic DNA was extracted with DNeasy Blood and Tissue Kit (Qiagen) from complete bodies or thorax parts of adults. In cases when yield was below 20 ng / µl, the extractions were concentrated by precipitation with sodium acetate according to the standard procedure and the precipitate was solubilized in distilled water in one-fourth of the original elution volume. A fragment of cytochrome c oxidase subunit I (COI) gene was amplified using Applied Biosystems 2720 Thermal Cycler. Initially, the C.myrtilli specific primers HybCamyCO (forward) and HybymaCCO (reverse) were used and PCR reactions were carried out as described by Nokkala et al. (2015) (link). Later on, to check the sequence at 5’ and 3’ ends, flanking primers HybCacoCO 5’-T7Promoter(F)-CTAACCATAARACTATTGGAAC-3’ (specifically designed forward primer) and a modified HCO (Folmer et al. 1994 (link)) reverse primer HybHCOMod 5’-T3-TAAACTTCAGGGTGACAAAAAATCA-3’ were used. PCR products were purified with QIAquick PCR Purfication Kit (Qiagen) and sequenced by Macrogen Europe (Amsterdam, the Netherlands). The sequences were trimmed to span a 638 bp stretch of the gene to match the available sequences of related C.myrtilli (Nokkala et al. 2015 (link)). Sequences obtained during this study have been deposited in GenBank under the accession numbers MF978762-MF978766.

The DNA template used for in vitro protein synthesis reactions was a PCR product made by amplifying the Photinus pyralis luciferase gene. The primers were designed such that a T7 promoter followed by a bacterial Shine–Dalgarno sequence was added prior to the start codon and a T7 terminator sequence was added after the stop codon, as per the manufacturer's recommendations (New England BioLabs).The primer sequences are listed below, where the firefly luciferase sequence (lower case letters), the T7 promoter (underlined), the Shine–Dalgarno sequence (bold) and the stop codon (italics) are indicated. Forward: 5′-GCGAATTAATACGACTCACTATAGGGCTTAAGTATAAGGAGGAAAAAATatggaagacgccaaaaacat-3′. Reverse: 5′-AAACCCCTCCGTTTAGAGAGGGGTTATGCTAGTTAcacggcgatctttccgccct-3′. Prior to use in coupled in vitro transcription–translation reactions, the PCR product was purified using the QIAquick PCR Purfication Kit (Qiagen) and resuspended in 0.5 × TE buffer to a final concentration of 50 ng μl⁻¹.