How can I quickly check if the cloning of my oligo into a backbone likePX330worked?
You can quickly check which of your colonies carry the correct insert (gRNA) inpX330/pX335with BbsI/AgeI double digestion. As a successful insertion will destroy the BbsI sites, a double digest should discriminate between positive and negative clones. Clones with insertion will show only linearized plasmid of ~8.5 kb (only AgeI will be able to cut). Clones without insertion will show a ~1kb and ~7.5kb fragment (both BbsI and AgeI will be able to cut).
https://www.addgene.org/crispr/zhang/faq/
AgeIでなくてもベクターを切って判別しやすい、BbsIのバッファーと相性がいいものを使えばいいんじゃない。入ったものはBbsIで切れないからそれで判別するということのようだから。 |
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