From my limited knowledge of science, I know transformation can be one of the hardest step in cloning, and that a large amount of research/trial and error has been done to improve on this step. I've heard electroporation is the most efficient technique, though I have no reference, and of course the cell, media and duration all have vital parts to play in the process.
However, I've never heard of supercoiling as an important factor. Size of the plasmid plays a large part in transformation, so intuitively it makes me think supercoiling the plasmid prior to transformation would improve the efficiency. I thought transformation was simply DNA diffusing through holes, but are parts of the plasmid required to bind/interact with the host?
Why aren't we suggested we supercoil prior to transformation? I have several thoughts but I'm just guessing:
- Is it because gyrase can disrupt the integrity of DNA?
- Or is it that it's just an expensive enzyme and the gain isn't worth it?
- Or perhaps supercoiling reduces efficiency of subsequent plasmid preps/transcription (though wouldn't the host's natural enzymes supercoil anyway?)