I am trying to clone with a pMAL-C2X vector carrying BamHI and SalI sites. I performed several times the ligation with the PCR insert and found several colonies. But seems all are empty vectors. To avoid confusion about the presence of an undigested plasmid, I made a ligation with digested vector without PCR plasmid and a digested plasmid without ligation then transferred to DH5alpha. Here I got colonies in the first case but no colonies in the latter approach. Apparently, I feel like there might be an occurrence of auto-ligation. is it possible to happen auto-ligation even if they have 2 different sites? Does anyone have experience with pMAL-C2X?
Restriction enzymes are site specific, but not ligases. So, there is a chance of self-ligation even though the vector is double digested.
There are two possible reasons why you can get self ligation with a double digested plasmid. First is that the stuffer fragment might act as insert in the ligation reaction giving you back the original plasmid. The second is that some fraction (even a tiny fraction) of the plasmid might not have been cut with one of the two enzymes, so this tiny fraction can readily recircularize itself. These are why the vector only religation is an important control to know the background in a reaction.
Did you confirm via sequencing that your isolated plasmid is identical to your original plasmid? Since you get no colonies from the non-ligated control, I would also assume that your vector re-circulizes with the fragment that you cut out. You could rule this out by either running the digested plasmid on a gel and cutting out the correct fragment, or by dephosphorylating the backbone so that your phosphorylated insert is necessary for circulization.
Maybe double check that both of your restriction enzymes are working properly. Also, I would consider running a colony PCR to quickly screen many clones for the presence of your insert. In the end, you only need that one good clone!
Good luck!
-Jan
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