Pcr4 topo ta cloning 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.

Each target locus of the ECFP gene was PCR-amplified from genomic DNA of individual adults (G₀) or pools of 5 to 20 G₁ or G₂ larvae. PCR products were either sequenced directly or cloned into a plasmid vector using the pCR4-TOPO TA cloning kit (Invitrogen, Life Technologies) prior to sequencing. From each Escherichia coli transformation, at least 10 colonies were picked and prepared for sequencing at the University of Missouri DNA Core. Mutated alleles were identified by sequence comparison with the original ECFP nucleotide sequence.

Onthophagus orthologs of candidate genes were identified by reciprocal BLAST to Tribolium and Drosophila databases. Corresponding DNA sequences were retrieved from existing Onthophagus genomic as well as transcriptomic databases. DNA fragments of a subset of candidate genes were then amplified through polymerase chain reaction (PCR) from existing complimentary DNA (cDNA) libraries (see Additional file 13: Table S6 for primer sequences), cloned into pCR 4-TOPO vector (pCR 4-TOPO-TA cloning kit, Invitrogen), and confirmed by resequencing. DNA fragments of the remaining candidate genes were synthesized from gBlocks Gene Fragments (Integrated DNA Technologies).