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Cloning using plasmid

  1. Mar 9, 2010 #1
    1. The problem statement, all variables and given/known data
    I have a problem that involves a plasmid DNA. The problem gives a plasmid diagram with the restriction sites and specifically tells me which restriction site the gene of interest would go. However, the restriction site is shown to be overlapping with the AmpR gene. Wouldn't that mean the Ampicillin resistance is deactivated when the restriction enzyme cuts the site? I don't get how the gene is supposed to be amplified in bacteria if the plasmid doesn't have AmpR...

    2. Relevant equations
    Biology - none

    3. The attempt at a solution
    See above
  2. jcsd
  3. Mar 9, 2010 #2
    I think the process you are reffering to is replica plating. It allows you to keep the colonies that you have produced in the experiment, whilst checking for certain genotypes. Some bacteria may take up the plasmid but not the recombinant form containing a gene of interest, by placing it within an antibiotic resistance gene, it inactivates that gene. There will be other resistant genes also present on the plasmid, and yopu can check the colonies against a number of antibiotics; this two step process allows you to distinguish between bacteria that have taken up the plasmid from those that haven't, determined by a functioning resistance gene on the plasmid that is not disrupted by restriction endonucleases; and then the colonies formed from the bacteria of interest (those with a plasmid that have incorprated a gene of interest) can then be distingiushed from those that have taken up a plasmid but do not contain the gene of interest, as those that have not taken up the gene of interest will be resistant to the second type of antibiotic, but the those of interest will not be resistant. Remember, replica plating does not destroy the colony of bacteria!
    I think this is right, I hope it helps.
    Last edited: Mar 9, 2010
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