2.2 DNA extraction, PCR and sequencing
Total DNA was extracted from up to 25 mg mantle muscle tissue using the
DNeasy Mini Kit (Qiagen, Germany) according to the standard tissue
protocol but reducing the final elution volume to 100μl. In order to
obtain mantle tissue, dissection was carried out using sterilized
forceps and scalpels, isolating the mantle from the rest of the tissues
such as intestine and tunic.
Cytochrome c oxidase subunit I (COI) PCR. The tunicate primers
pair Tun_reverse2 (Rev) (Stefaniak et al., 2009) and Cve-CO1-F54 (Fwd)
5´ AGTGTTTTAATTCGAACAGA 3´, and the primers pair Deg COI F2 (Fwd) and
Deg COI R2 (Rev) (Reem, Douek, Paz, Katzir, & Rinkevich, 2017) were
used for amplification. The primer Cve-CO1-F54 (Fwd) was designed within
this study as consequence of bad quality (double peaks, ill-defined or
garbled peaks in the chromatograms) forward sequences obtained with the
Stefaniak-primer, our primer was designed using the software Geneious
version R8 (Kearse et al., 2012), and based on good quality forward
sequences from this work. Reactions were carried out in 25 μl volumes,
using 0.025 U/µl of Promega GoTaq G2 Flexi DNA Polymerase, 30 ng of DNA,
0.5 µM of each primer and 2 mM of MgCl2. The
amplification protocol was 2 min at 94°C for initial denaturation
followed by 36 cycles of 60 s at 94°C, 50 s at 46°C, 50 s at 72°C, and a
final elongation step of 8 min at 72°C.
Nuclear Ribosomal RNA Gene (18S rDNA) PCR. Primers 18S1 (Fwd) and
18S4 (Rev) (Tsagkogeorga et al., 2009) were used for amplification.
Reactions were carried out in 25 μl volumes, using 0.03 U/µl of TaKaRa
LA Taq HS, 30 ng of DNA, 0.5 µM of each primer and 0.05 mM of Betaine.
The amplification protocol was 1 min at 94°C for initial denaturation
followed by 30 cycles of 10 s at 98°C, 50 s at 50°C, 2 min at 72°C, and
a final elongation step of 10 min at 72°C.
PCR products were visualized on a 1 % TAE agarose gel stained with
GelRed (Nucleic Acid Gel Stain) under UV illumination. PCR products were
outsourced for sequencing to Eurofins MWG Operon (Germany) on an
ABI3730XL automatic DNA sequencer, using either of the two primers used
for amplification.