Low transformation efficiency with PX459 CRISPR vector?

I ran into trouble with transforming competent e.coli with my PX495-based construct. I'm using is pSpCas9(BB)-2A-Puro (PX459) V2.0 from Feng Zhang's MIT lab ( https://www.addgene.org/62988/ ) as a backbone vector and XL10-Gold cells for cloning. However, XL10 produced no colonies after plating with ampicillin, and DH10 only produced a couple of colonies with a very low DNA yield after isolation with a Quiagen Miniprep kit. I know this sounds basic, but could anyone please advise any strategies to troubleshoot this?


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Maroon Basiliskalmost 5 years ago

I'm having the same problem here. Did you manage to solve it?

Amethyst Dragonalmost 5 years ago

Home made Top10 competent cell gave me whole plate colonies and most of them are correct after sequencing.

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