2.2 | Primer design
Initial ctenophore COI primers were designed based on COIsequences from our unpublished ctenophore transcriptome data, published
mitochondrial sequences of Mnemiopsis leidyi (GenBank accession
#: NC016117, JF760210), Pleurobrachia pileus (GenBank accession
#: JF760211), and the ctenophores which we could successfully amplify
with ‘Folmer primers’ (Folmer et al. 1994) (Figure 1). RNA
extractions, transcriptome sequencing, and analyses were conducted as
previously described (Francis et al. 2013). We designed primers
for the mitochondrial COI gene with Primer3 (Untergasser et
al. 2012) within Geneious Prime (v. 2020.2.3, www.geneious.com), using
PrimerQuest (www.idtdna.com), or by eye based on nucleotide alignments.
Primer design was an iterative process, with multiplexed PCR assays
suggesting which variants and combinations of forward and reverse
primers worked best. Primer positions were numbered relative to the
sites as they occur in M. leidyi (JF760210; Pett et al.2011), as indicated in Figure 1.