Does anyone have any experience cloning inserts into the Pichia expression
vector pHIL-D2? I've been trying to clone an EcoRI PCR fragment (~700 bp)
into EcoRI-digested, alkaline phosphatase-treated pHIL-D2 (8.2 kb) without
success so far. I realize that cloning fragments into large vectors is less
efficient than smaller vectors but I don't feel I should be having the
problems I am having with a simple stick-end ligation. Comments or suggestions?