T7 t3 megascript kit

The sequence for Tlx transcript was recovered from a newly assembled transcriptome of P. carnea. Tlx was amplified from medusae cDNA using the following PCR primers: P. carnea forward 5´- GAAAGATAAACACGAAAAAGAAACGG-3´ and reverse 5´-TCCGGAACTTCATTACTCGCTGTTGC-3´ for an expected amplicon length of 528 bp. Amplicons were cloned using the Invitrogen pCR4-TOPO-TA Cloning Kit and sequenced using M13 forward and reverse primers. Sense and antisense DIG labeled riboprobes were synthesized from clones using the Invitrogen T7/T3 Megascript kit. In situ hybridization (ISH) protocol was adapted from ref. ^{56 (link)}. Only the fixing solution, the amount of probes and the alkaline phosphatase signal detection solution were changed from the original protocols and are described below. Animals were fixed in ice cold fix (3.7%PFA and 0.25% glutaraldehyde in 1X PBS). Hybridization was carried out at 50 °C for 18 h with a probe concentration of 1 ng/µl. DIG labeled riboprobes localization was detected by immunostaining with anti-DIG-Fab-AP (ROCHE) and NBT/BCIP.

Several polyp-specific transcripts identified during the DE analyses were selected for confirmation and further investigation with whole mount in situ hybridization (ISH) experiments (Table 3). Sequences for these transcripts were identified in the assembly, amplified from cDNA, cloned using the Invitrogen TOPO-TA Cloning Kit, and anti-DIG labeled riboprobes were synthesized from clones using the Invitrogen T7/T3 Megascript kit. ISH of these transcripts were performed following methods from Nawrocki & Cartwright [76 (link)].