8
$\begingroup$

All the examples on DNA cloning I have encountered have assumed that the target gene and vector both have compatible restriction sites at just the right locations (probably for ease of explanation). Instead suppose I have a vector that has restriction sites for multiple restriction enzymes, but my target DNA sequence doesn’t have a restriction site for any of them, how would I go about creating a recombinant DNA from these?

$\endgroup$
1
  • 2
    $\begingroup$ You could use a guide RNA and CRISPR/CAS for any sequence. However, the accepted answer is cheaper and more straightforward if you don't mind a slightly altered sequence (typically, a silent mutation or a change outside of the region of interest). $\endgroup$ Mar 29 at 15:39
11
$\begingroup$

If you're synthesizing the insert then you just design it with restriction sites that are compatible to the vector. Otherwise, you can design PCR primers that have restriction sites in them. That way when you amplify your insert you'll attach the restriction sites to it. EMBL, Addgene, and NEB all offer somewhat more detailed explanations of this on their sites.

$\endgroup$

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

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