PlasmidSafe vs NEB Endonuclease V

[Erik Nielson]

Hi All,

Has anyone tried replacing the PlasmidSafe for NEB RecBCD Endonuclease V. I was having mediocre results with PlasmidSafe for hexamer production and ran out, ordering things through NEB works better for our lab so ordered their product RecBCD Endonuclease V.

It has identical concentrations 10U/ul to PlasmidSafe.

Has anyone use this in their work? Can you describe any troubleshooting steps you undertook?

Thanks,

Erik Nielson.

[Erik Nielson]

If anyone is curious utilizing the neb endonuclease appears to work. I have been able to successfully amplify hexamers from digests using NEB RecBCD with about the same rate as PlasmidSafe.

I have also found that increasing the heat inactivation time from 30min to 1 hr (75 C) of the nuclease increases my success rate as well.

Additionally Herc II pol seems to work in annealing temperatures between 60 and 64 C, anything higher has dramatic drop off of activity. 

Good luck with your experiments,

Erik.

[pdh...@gmail.com]

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