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Can DNA ligase seal two non-complementary sticky ends if the reaction is incubated at the optimum conditions ? I do almost 100% double digestion in the lab. However, during ligation, when I do a control without the insert, I still get many circularized vector and they cause massive false positives during transformation. Can ligase sealing two non-complementary sticky ends be a possible reason behind this ?

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  • $\begingroup$ Since DNA ligase can ligate blunt cut DNA, it can probably ligate incorrect sticky ends, but the probability is low. Are you doing an antarctic phosphatase reaction to remove the 5' phosphate on your vector? That would reduce the liklihood of bad ligations. $\endgroup$ – user137 Feb 16 '15 at 23:39
  • $\begingroup$ Unfortunately I am not doing that because some people can make cloning work without phosphatase treatment in our lab and I am mostly following their procedures. At least so far. $\endgroup$ – ecagl Feb 17 '15 at 0:02
  • $\begingroup$ I'd definitely try a phosphatase control and see if that's the issue. $\endgroup$ – MattDMo Feb 17 '15 at 2:16
  • $\begingroup$ You may be getting false positives because of incomplete digestion too. Did you check the digested plasmid on the gel? Also use very little of the plasmid (≤500ng) in ligation $\endgroup$ – WYSIWYG Feb 17 '15 at 4:47
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    $\begingroup$ They found here the T4 Ligase can effectively ligate mismatched ends resulting in up to 5 mismatched bases. Which ligase are you using? $\endgroup$ – CKM Feb 17 '15 at 23:20
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If I understand correctly you are preparing your cloning vector by digesting it with two enzymes that are 31 bp apart. If you have a high background of recircularized vector see if there is a unique restriction enzyme site within that 31 bp. If there is, and if the fragment you are trying to insert also lacks that site, then a simple approach would be to inactivate the ligase, and digest the reaction with the third enzyme. That should drastically reduce the background of recircularized vector. If after gel purifying the vector backbone after the double-digestion, you get a significant background of recircularization, then you can easily test your hypothesis about ligase ligating mismatched sticky ends: both of the starting sites should be gone and any sites in-between them should be gone. Have you tested this?

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    $\begingroup$ I have solved the problem by increasing the time of digestion and preparing new plates. Now I get only a few colonies when there is no insert - probably there are still some single digests and ligase repairs the nick- and it is not a major problem anymore. I have read about your suggestion elsewhere but there was only one such site between my sites and the enzyme for that was rare & expensive so I followed another approach as I mentioned. Apparently I got false positives because my enzymes were sloppy and my plates were a bit old. $\endgroup$ – ecagl Mar 22 '15 at 21:51

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