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This is as far as my understanding of gene cloning goes. Let's say that the plasimd (vector) in the bacteria contains an ampicillin resistence gene. Through restriction enzymes, the ampicillin resistance gene is cut through and foreign DNA is inserted. This creates a recombinant DNA molecule.

Once many bacteria are introduced to a medium that contains ampicillin, only ones that have no recombinant DNA and functional ampicillin resistance genes will survive and those with the recombinant DNA won't. Through the addition of some chemicals, the surviving bacteria will turn blue and those with the recombinant DNA will turn white. Thus, bacteria with recombinant DNA can be taken and grown in their own culture, the recombinant DNA can be isolated, and research goes on.

However, if all the bacteria with recombinant DNA have been killed because they have no functional ampicillin resistance genes, how can they be 'grown' in their own colony? They're all already dead, aren't they?

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  • $\begingroup$ This topic has been covered in previous questions, e.g. biology.stackexchange.com/questions/69141/…. $\endgroup$ – David Feb 17 at 22:03
  • $\begingroup$ You are mixing up too many things: 1) the ampicilin gene is normally not cut out to make recombinant DNA. Instead 2) cutting/pasting into a vector is usually performed at a multiple cloning site (MCS) independently of the ressitance gene. 3) The blue/white screening uses a beta-lactamase gene and is also unrelated to ampicilin. $\endgroup$ – Nicolai Feb 18 at 9:40
  • $\begingroup$ @Nicolai yep I realized how off it was and answered my own question. I would've deleted the question because of how confusing it was but wasn't able to because it already had an answer. $\endgroup$ – Korvexius Feb 18 at 16:48
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I’ll elaborate on this answer in the future but, in the meantime, blue/white screening is used to differentiate between bacteria that were transformed with the vector and gene of interest and bacteria that were transformed with an empty vector. An empty vector still confers antibiotic resistance and thus allows the bacteria to grow, but it does not contain the gene of interest and is therefore useless to the researcher.

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The question confuses too many things. The process being described, Lac Z Blue-White Screening, doesn't involve cutting the ampicillin gene, nor do the bacteria with the recombinant DNA or without the recombinant DNA die in the process of the experiment.

The plasmid contains a Lac Z gene involved in creating enzymes that break down galactose. In a test tube, the vector plasmids take up the gene of interest and the recombinant DNA is produced. The recombinant DNA is then added to the medium of bacteria, but only some of the bacteria will be transformed and take up the plasmid vector. In these bacteria, restriction enzymes will 'cut open' a space for insertion in the Lac Z gene, and DNA ligase will seal it up. The insertion of the gene in a bacterium disrupts the Lac Z gene and make it unable to produce the required enzymes to break down galactose. Then, the bacteria are applied to a Petri dish that contains growth media with a derivative of galactose, X-gal in it. When X-gal is broken down by the bacteria able to create the necessary enzymes from their functional Lac Z genes, the bacterial colony will turn blue. So, when you add these bacteria into the Petri dish, only the bacteria without the recombinant DNA will break down X-gal and turn blue, whereas bacteria with the recombinant DNA will not be able to break it down and remain white. This allows the researcher to distinguish between the bacteria with and without the recombinant DNA, and allow them to isolate for the ones with it.

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  • $\begingroup$ I answered my own question because I learned in the time since I asked it just how off it was. $\endgroup$ – Korvexius Feb 18 at 16:47
  • $\begingroup$ Your description of how blue/white staining works is (essentially) correct, however the generation of recombinant DNA does not happen in bacteria. Recombinant plasmids are generated by researchers in vitro (meaning in a test tube) using restriction enzymes (and more like DNA ligase). Note that in most cases even a plasmid that did not receive the gene of interest would still be recombinant, since the even the base plasmid used is usually not found in that form in the bacteria used. $\endgroup$ – Nicolai Feb 18 at 16:56
  • $\begingroup$ @Nicolai I've updated the answer given your remarks. Do you think it's good now? $\endgroup$ – Korvexius Feb 19 at 0:26

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