I have a pSUPER retor puro vector that already has a cloned shRNA. I wanted to remove the already cloned shRNA and replace it with the other shRNA. the user manual from oligoengine sys that upon cloning between bgl II and Hind III, the Bgl II site is modified to Bam HI. I was wondering if I could remove the already existing shRNA by the other one using Bam HI and Hind III digestion. Is it possible?
It is possible to replace using careful selection of restriction sites. However, isolating pure colonies that has only the desired shRNA is tricky. Often you may have a mixture of these two. So, I would recommend an empty pSUPER to start with. No hassles..!!
thank you. I was curious to know why shRNA cloning is so tricky? earlier I faced problems in cloning the annealed olgos, then with screening and now with getting proper plasmid concentration after isolation.. why is it so tricky?
Anneal sticky end shRNAs for ease. colony PCR should speed up screening process. plasmid concentration depends on copy number. Good Luck..!!
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