PhD thesis - Biologisk Institut - Københavns Universitet

19 Material and methodsβ-Actin F1 5’-TGGGAYGAYATGGARAARATβ-Actin R1 5’-GCCATYTCYTGYTCRAAα-Tubulin (Zheng et a. 1998)AT16-F 5’-ATHGGHAAYGCNTGYTGGGAT412-R 5’-RAAYTCDCCYTCYTCCATDCCβ-Tubulin (Garant and MacRea 2009)BT-F 5’-GGNCARTCNGGNGCNGGNAAYAAYTGGGCNBT-R 5’-NGGRAANGGNACCATRTTNACNGC50 °C50 °C55 °CRapid amplification of cDNA ends (RACE)Fragments of Themiste pyroides homeobox containing genes were isolated bydegenerate PCR using cDNA of mixed larval stages as template. In addition, 5’ and 3’cDNA were generated using the same template for rapid amplification of cDNA ends(RACE) with the SMARTer RACE cDNA amplification kit (Clonetech,Mountainview, CA, USA).For each of the recovered gene fragments, which ranged between 135 and 271base pairs (bp) (Table 3), gene specific primers (GSP) were designed and used forRACE (Advantage 2 PCR Kit; Clonetech). Touchdown PCRs were performed withfollowing amplification parameters: 5 min at 94 °C, 10 cycles of 45 sec at 94 °C, 45sec at 57 °C (every cycle -1 °C), 3 min at 72 °C, and 25 cycles of 45 sec at 94 °C, 45sec at 47 °C, and final 30 sec extension at 72 °C. Reactions were agarose-gelelectrophoresed, stained with SYBRsafe (Invitrogen, Taastrup, Denmark), andphotographed under UV light. Subsequently, 1 µl of RACE PCR products were usedas a template for another RACE PCR with nested GSPs (see Table 3). The parameterswere: 5 min at 94 °C, 35 cycles of 45 sec at 94 °C, 45 sec at 60 °C, 30 sec at 72 °C,and a final 10 min extension at 72 °C. Positive fragments were purified from theagarose gel, cloned into the vector pGEM-T easy, and sequenced as described above.However, I was able to amplify into the 3’ direction of two genes only, hox1 (47 bp)and hox8 (73 bp).