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For a DNA fragment to be inserted, it must have two restriction sites on either end. My textbook makes it seem that this is naturally occurring but it seems a little too convenient, are they added in instead? And also, are promoter and terminator regions already present in the fragment or is the restriction site separated and reattached in order to insert them?

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    $\begingroup$ Depends on what you're trying to do, what materials you have to work with. What are you trying to do? What textbook are you using? What websites have you looked at? $\endgroup$
    – Armand
    Oct 5 '21 at 2:53
  • $\begingroup$ I’m an A level student, using an aqa textbook. I’ve tried looking it up but the answers are over my head. I’m just trying to understand whether the restriction sites on the DNA fragments are added in sometimes rather than always naturally occurring. $\endgroup$
    – Pizaaaa
    Oct 5 '21 at 7:05
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    $\begingroup$ Welcome to the site. Please visit the help center and take a tour. In particular pay attention to the homework page. For these we require evidence that you have put some effort into working out the answer yourself. $\endgroup$
    – bob1
    Oct 5 '21 at 7:56
  • $\begingroup$ I suggest you look up plasmids and have a play with the maps and sequence. Try Addgene for this. $\endgroup$
    – bob1
    Oct 5 '21 at 8:05
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That depends a bit on the method you are using, but mainly you are right, it is rather unlikely that a piece of DNA contains the right restriction site on both ends, let alone even two different ones for site directed cloning.

A common technique used to insert the restriction sites of choice is to amplify your DNA with PCR and special primers. These primers contain your wanted restriction sites next to the recognition sites and produce an overhang (at least in the beginning of the amplification reaction) which contains the restriction site. See the image (from here) on how this technology works.

enter image description here

Regarding promoter and terminator regions, I assume that you clone something into a DNA expression vector. Then these sequences are typically part of the vector and optimized for the cells to work with (bacterial or eukariotic).

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  • $\begingroup$ thank you! that makes sense $\endgroup$
    – Pizaaaa
    Oct 5 '21 at 10:40

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