I amplified a 2,976 bp sequence so that I could ligate it into a plasmid of approximately 3.6 kb, but when I ligate and then perform the bacterial transformation I only get the plasmid ligation without in insert.
I have already checked that the restriction enzymes are digesting correctly.
I think the size of the insert might affect the ligation, so I am looking for a protocol to improve the ligation efficiency between the plasmid and the insert.
If you use a single restriction site for cloning, self-ligation leading to recirecularization of the plasmid will be far more frequent than the incorporation of the desired insert. You can improve this by using two different restriction enzymes that generate non-compatible ends. Most vectors make this possible, since they feature a multiple cloning site (MCS) with a variety of different unique restriction sites. Nevertheless, recircularization can be a problem if the double digestion of the plasmid is not complete. It is possible to further decrease the background of empty vector by treating the ligation mixture with a restriction enzyme whose site lies between the two sites of the MCS chosen for cloning, provided that the insert contains no such site, of course.
You can also phosphatase treat your vector (not the insert!) to reduce self-ligations. A lower temp and longer time may help too.
I've been trying to do something very similar (insert a 3.5kb insert into a 10kb plasmid). You need to get an appropriate ratio of vector to insert (1:3 is the standard); I'd recommend doing one ligation reaction with that ratio and another couple at different ratios (1:10 and 10:1 for instance) and a control containing just vector, ligase and ligation buffer. With Invitrogen T4 ligase and ligation buffer, I've had successful insertions after 4 hours ligating at room temperature followed by transformation.
NEBioCalculator (https://nebiocalculator.neb.com/#!/ligation) if you haven't come across it before, is a good tool for giving you ratios of insert to vector to put in your ligation mix.
Desphorylation of the vector (NOT the insert) is also a good idea, although not really necessary if you're using two different restriction enzymes to cut into your plasmid and insert your sequence.
Amy Klocko Khajezade Mohadese Pierre Béguin Hugh White thank you all very much for your help, I will follow the recommendations you have left me.
Hello Brandon, what vector are you using please? And in case you have any issue with the vector, try one vector I am currently using. It worked well for me. It is a ligation independent vector_ pET30a. Thank you.