Advantage 2 taq polymerase

Zebrafish Ikk1 (Chuk) cDNA was purchased from Open Biosystems (Clone ID: 5914247, GE Dharmacon). Full-length (FL)-Ikk1 was amplified with Advantage 2 Taq Polymerase (Clontech) using forward primer 5′-GCTAGCTGTCAATATGGAGAAACCCCCT-3′ and reverse primer 5′-TAATGGATTTGGTACCGACAAACGCGCTGATTTA-3′. The amplified PCR products were cloned into pCR Blunt II-TOPO using the Zero Blunt^® TOPO^® PCR Cloning Kit (Life Technologies). To generate tdTomato fusion constructs, pCRTOPO-FL-Ikk1 and ptdTomato-N1 vector (Clontech) were digested with KpnI and NheI, and ligated. The Ikk1(C179A)–tdTomato variant was generated from FL-Ikk1–tdTomato using site-directed mutagenesis (GeneArt^® Site-Directed Mutagenesis PLUS Kit, LifeTechnologies). The constructs were subsequently cloned into a plasmid containing the zebrafish tp63 promoter (pT2KXIG_tp63:AcGFP, courtesy of Gromoslav Smolen, Harvard University, Cambridge, MA). First, GFP was removed via digestion with BamHI and NotI, and both sites were Klenow blunt-ended. The Ikk1–tdTomato plasmids were digested with SnaBI and SfoI and ligated into pT2KXIG_tp63 to generate tp63:FL-ikk1–tdTomato and tp63:ikk1(C179A)–tdTomato. HEK001 were transfected using Nanofectin (PAA, Q050-005) when 60% confluent in 8-chamber vessels. It was determined that 0.5 µg of DNA per 0.6 µl of transfection reagent was optimal.

Degenerate primers were designed based upon multi-species alignments (GenBank). Briefly, degenerate primer pairs developed for the mussel were used on cDNA from three randomly selected mussel samples. Degenerate primer pairs were designed to amplify 14 genes of interest and one ribosomal housekeeping gene (Table 4). The PCR amplifications using these primers were performed on 20 ng of each cDNA sample in 50 µl volumes containing 20–60 pmol of each primer, 40 mM Tris-KOH (pH 8.3), 15 mM KOAc, 3.5 mM Mg (OAc)₂, 3.75 µg/ml bovine serum albumin (BSA), 0.005% Tween-20, 0.005% Nonidet-P40, 200 µM each dNTP, and 5U of Advantage^® 2 Taq polymerase (Clontech, Palo Alto, CA, USA). The PCR was performed on an MJ Research PTC-200 thermal cycler (MJ Research, Watertown, MA, USA) and consisted of 1 cycle at 94 °C for 3 min, and then 40 cycles at 94 °C for 30 s, at 60 ° C for 30 s, and 72 °C for 2 min, with a final extension step of 72 °C for 10 min. The products of these reactions were electrophoresed on 1.5% agarose gels and resulting bands visualized by ethidium bromide staining. Definitive bands representing PCR products of a predicted base pair size of the targeted gene were excised from the gel, and extracted and purified using a commercially available nucleic acid-binding resin (Qiaex II Gel extraction kit; Qiagen, Valencia, CA, USA).

Degenerate primers were designed based upon multi-species alignments (GenBank). Briefly, degenerate primer pairs developed for the razor clam were used on cDNA from three randomly selected razor clam samples. Degenerate primer pairs were designed to amplify five genes of interest and two ribosomal housekeeping genes (Tables 2 and 3). The PCR amplifications using these primers were performed on 20 ng of each cDNA sample in 50 μl volumes containing 20–60 pmol of each primer, 40 mM Tris-KOH (pH 8.3), 15 mM KOAc, 3.5 mM Mg (OAc)2, 3.75 μg ml⁻¹ bovine serum albumin (BSA), 0.005% Tween-20, 0.005% Nonidet-P40, 200 μM each dNTP, and 5U of Advantage^® 2 Taq polymerase (Clontech, Palo Alto, CA, USA). The PCR was performed on an MJ Research PTC-200 thermal cycler (MJ Research, Watertown, MA, USA) and consisted of 1 cycle at 94 °C for 3 min, and then 40 cycles at 94 °C for 30 s, at 60 °C for 30 s, and 72 °C for 2 min, with a final extension step of 72 °C for 10 min. The products of these reactions were electrophoresed on 1.5% agarose gels and resulting bands visualized by ethidium bromide staining. Definitive bands representing PCR products of a predicted base pair size of the targeted gene were excised from the gel, and extracted and purified using a commercially available nucleic acid-binding resin (QIAEX II Gel extraction kit; Qiagen, Valencia, CA, USA).