Take the 2-minute tour ×
Biology Stack Exchange is a question and answer site for biology researchers, academics, and students. It's 100% free, no registration required.

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!

share|improve this question
add comment

2 Answers

up vote 5 down vote accepted

Remember than Pvu1 cuts in the middle of AmpR, 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 TetR 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.

share|improve this answer
    
Thank made so much sense omg thank you!!!!!! –  Jesse Dec 16 '13 at 3:01
add comment

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

share|improve this answer
add comment

Your Answer

 
discard

By posting your answer, you agree to the privacy policy and terms of service.

Not the answer you're looking for? Browse other questions tagged or ask your own question.