Performed PCR to amplify the C.gigas hexokinase (ACCESSION#) promoter region (-2059bp) and the CDS without the stop codon. Elimination of the stop codon allows for subsequent cloning into the pBAD-TOPO expression vector, which will incorporate the V5 epitope tag sequence. This tag will be used to distinguish between endogenous hexokinase expression and expression generated from our hexokinase construct.
Used hexokinase primers (SR IDs: 1518, 1519).
Master mix calcs and cycling params are here.
Results:
Ladder: Hyperladder II (Bioline)
No amplification in sample or no-template control. Will re-do with lower annealing temp (50C).