pET Vector Cloning Issue

[sd_labber]

I’ve been trying to clone a few geness into pET19b using RE sites NdeI and BamHI. I first digest overnight with NdeI, heat inactivate at 65 degrees for 20min, then digest with BamHI for 4hr and CIP for 1hr before gel purifying on a 0.8% agarose gel. The gel always looks good when compared to digested and undigested control vectors (I see 2 bands at expected sizes for the linearized DNA).

For ligations I’ve been utilizing Quick Ligase with a 3:1 ratio of insert:vector. My controls have been the same as the ligations, minus insert, and only yield 1-2 colonies at the most, while my ligations have many (20-30) colonies per 50uL of C43 DE3 cells plated. However, when I pick colonies for minipreps, most of the colonies run lower than, or even with, the undigested pET vector. I’ve even digested the miniprepped samples (thinking maybe nicked v. supercoiled DNA maybe responsible for the difference) and found no insert.

Has anyone worked with pET vectors and encountered similar issues? I’d appreciate any advice, thanks!!!

[mmorgan]

Originally Posted by sd_labber

I’ve been trying to clone a few geness into pET19b using RE sites NdeI and BamHI. I first digest overnight with NdeI, heat inactivate at 65 degrees for 20min, then digest with BamHI for 4hr and CIP for 1hr before gel purifying on a 0.8% agarose gel. The gel always looks good when compared to digested and undigested control vectors (I see 2 bands at expected sizes for the linearized DNA).

For ligations I’ve been utilizing Quick Ligase with a 3:1 ratio of insert:vector. My controls have been the same as the ligations, minus insert, and only yield 1-2 colonies at the most, while my ligations have many (20-30) colonies per 50uL of C43 DE3 cells plated. However, when I pick colonies for minipreps, most of the colonies run lower than, or even with, the undigested pET vector. I’ve even digested the miniprepped samples (thinking maybe nicked v. supercoiled DNA maybe responsible for the difference) and found no insert.

Has anyone worked with pET vectors and encountered similar issues? I’d appreciate any advice, thanks!!!

It's possible the ends of your vector are getting a bit funky with the extensive treatment that you do. You don't need to heat inactivate the NdeI, just go into high salt buffer with BamHI. Also leave out the CIP step it's not necessary. I would try: 3 hours NdeI, then add Buffer 3 (high salt) and BamHI for 3 hours. Gel purify and then use the plasmid.

Usually when I get strange cloning products like the ones you describe it's because the ends of the vector become damaged. It is odd that you don't get higher background though, this suggests something in your insert sample is ligating into the plasmid (contaminating primer-dimers maybe???).

Also are you cloning in a protein expression strain (C43 DE3) ??? This is not recommended because these strains often have high mutation rates. Best to use something tried-and-true like DH5-alpha.