4 replies to this topic

[fzhang]

hi, everyone. If the ligation efficiency is so low that the majority of the plasmid vectors remain in the linear conformation, can they be transformed into the competent cells?

And if so, can these linear vectors express the antibiotics resistance genes on them?

Thanks a lot.

[tfitzwater]

Many years ago I transformed HinD III-linearized plasmid in the absence of ligase by CaCl2 into C600 cells as a control and got hundreds of transformants on amp plates. These were completely eliminated by dephosphorylating the linear fragments before transformation.

Edited by tfitzwater, 14 October 2009 - 07:05 AM.

[Flyingbike]

I think only ligated (circular) vectors are able to express in cells, in theory

[aleruiz]

Once I read that competent E. coli can only be transformed with non-linear (circular) plasmids, because if a linear plasmid enters to the cell, nucleases attacks DNA. It's suppose to be different in eukaryotic cells, these can be transfected with linear plasmids and express all stuff in there.

[tfitzwater]

The C600 cells mentioned in my previous post are a strain of E. coli. Therefore, yes, linear plasmids can be transformed into E. coli and selected by ampicillin resistance. Plasmid preps of the colonies indicated that the linear DNA had been converted into normal supercoiled DNA. Dephosphorylating the linear fragments prior to transformation prevented them from circularizing and prevented colonies (also proving that my gel-purified HinD III-cut DNA was not contaminated with circular DNA).

C600: F-, e14, mcrA, thr-1 supE44, thi-1, leuB6, lacY1, tonA21, hsdR-, hsdM+, mcrA-, mcrB+