Which restriction enzyme would i use?

Question

Plasmid pBr322 includes two genes that confer antibiotic resistance: a gene for ampicillin and a gene for tetracycline. The cutting site for the restriction enzyme BamH1 is in the middle pf the tetracycline resistance gene. The cutting site for the restriction enzyme Pvu1 is in the middle of the ampicillin resistance gene.

If you were using a cell culture plate that contained ampicillin, which restriction enzyme (BamH1 or Pvu1) would you use to cut the plasmid and excise the gene you wanted to insert?

Attempt:

I would assume you need to use Pvu1 since the cutting site is in the ampicillin resistance gene, but the correct answer is BamH1. Why?

Any help would be awesome! Thanks!

Answer 1

Remember than Pvu1 cuts in the middle of Amp^R, and when you insert your gene you'll be disrupting its function, meaning that any transformed colonies will no longer be able to grow on Amp-containing media. This is why you want to cut with BamH1 - the disruption to the Tet^R gene is irrelevant, and it leaves Amp resistance in place.

The point of growing on antibiotic-containing media is to select for cells that have taken up your plasmid - cells that haven't can't grow.

Answer 2

I would go with BamH1 enzyme as the cut site in pBr322 is BamHI 375 and ss you are plating it in a ampicillin plate you need the ampicillin resistant gene to be intact for screening so by using BamHI you will not disrupt the Amp resistant gene