Cloning into LentiCRISPRv2 plasmid

Is it possible to digest a LentiCRISPRv2 plasmid (with a sgRNA cloned in and not the the 2kb filler) using BsmBI? I have done it before and it worked well, but I don't see the BsmBI cut sites after sequencing with the hU6 promoter. I don't think the BsmBI cut site is destroyed during the cloning process.


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Rose Mermaidover 3 years ago

I don't actually think you can. It appears the recognition sites for BsmBI are inside the 2 kb filler, and only the overhangs are included in the sgRNAs. It's more likely that you had unligated cut plasmid remaining somehow.

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