Hello Someone Could you give me please information that permit me explicate Why I see two bands when I do PCR of genomic DNA of X-33 as the host but when I do PCR with genomic DNA of KM71H strain I see only one band. What is the reason for this happen?
I know that one band is the AOX1 gen 2,2Kb and the other band is product of add my insert and 588bp of vector (Ppicza), But I don't have explication why see two bands in a PCR product, where usually I see only one...
Hi there,
Stable transformation of Pichia is based on insertion of the full plasmid into the genome. The gene of interest is cloned between AOX1 promoter and AOX1 terminator sequences (just like the AOX1 gene). Then once insertion is completed, there are two different genes surrounded by AOX1 promoter and terminator sequences. The primers used for PCR screening are from AOX1 promoter and terminator therefore two régions of the genome may be amplified : AOX1 gene region and your favorite gene region which result into 2 bands on gel provided sizes are different enough.
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