RF-Cloning.org Forum

Alexander

Member

Registered: 2014-04-28

Posts: 1

Relevance of DpnI treatment?

Hi there,

I'm planning to do my first RF-cloning experiments, but I was wondering why the cloned plasmid is treated with DpnI at the end of the protocol...

Thanks!
Alexander.

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Relevance of DpnI treatment?

Hey Alexander,
I hope your experiment goes well!
The DpnI is crucial, because it chews up all the parental plasmid in the reaction. If you skip the DpnI step you'll end up with tons of transformants, but most of them will have no insert.
Also, you MUST grow up the plasmid in a dam+ strain of E. coli for DpnI to work (the standard sub-cloning strains, like DH5-α, generally fulfill this requirement).
-Steve

bjartelund

Member

Registered: 2014-01-23

Posts: 1

Re: Relevance of DpnI treatment?

We published a paper this spring vector containing ccdB-toxin  to select for transformants instead of using DpnI for selection.
Lund, B. A., Leiros, H.-K. S., & Bjerga, G. E. (2014). A high-throughput, restriction-free cloning and screening strategy based on ccdB-gene replacement. Microbial Cell Factories, 13(1), 38. doi:10.1186/1475-2859-13-38

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Relevance of DpnI treatment?

Thanks for sharing Bjarte!
It's a really cool use of ccdB, and looks like a great technique if you're swapping lots of inserts into a specific expression plasmid.
-Steve