I am planing a cloning experiment, but I have a hard time to see how I could manage this.
I have a reporter vector from Clontech (pLVX-DD-ZsGreen1 , pLVX-DD-ZsGreen1 Reporter.pdf 14.17KB 314 downloads), which has a PGK-puromycin cassette.
I want to replace puro by RFP. In recipient vector pLVX PGK-puro cassette could be excised by digestion with SphI and SexAI.
I have a vector (pBluscriptKS) which contains PGK-RFP, but in a 3' to 5' orientation ( pBS_mPGK_mRFP (4143bps).pdf 12.85KB 522 downloads):
XbaI - RFP - BamHI - PGK-promoter - (SphI) - EcoRI. (Why does this nevertheless lead to fluorescent cells when transfected?)
If was planning to add a SexAI site 5' to XbaI site via PCR. Then I would have SexAI(- RFP - PGK-Prom -)SphI which would be compatible to pLVX digested with SphI and SexAI.
The problem is now that the orientation of the insert is inverted. So as far as I managed to imagine it, this would lead to the interchange of sense and antisense strand, and therefore no transcription.
Is that correct? If yes I have to find another strategy, which I am not sure I will :-)
Thanks for answers.
Edited by loucash, 02 August 2010 - 04:37 AM.