Ligation problems - (Jul/19/2001 )
Hi! I am trying to perform a blunt end ligation! After Restriction enzyme digestion, I blunted the vector with T4 DNA pol before alkaline phosphatase treatment and Gene Clean purification. The insert is a PCR product that had been digested with enzymes creating blunt ends. But the ligation (using 5 U ligase) did not yield any plasmids having the expected length of vector and insert!Does anybody know what is going on here?
Thanks a lot!
I believe that klenow is the enzyme of choice when filling in blunt ends. Reaction conditions are provided in the catalogues for klenow.
Also be sure that you are not engineering your restriction sites directly on the ends of the primers for amplifying your insert.I always place 2 extra base pairs at the end. Sometimes the enzymes do not cut efficiently if they have to dock directly on the end of the DNA.
Hi! Try to treat cutted vector by AP (Alkaline Phosphatase). It prevents it from selfligation.Good luck.
You can use Klenow or T4 for blunt ends but it depends on the type of overhang, one works best for 3' and the other for 5'.Try using more ligase as blunt end cloning tends to need more, try 100fold more ligase!
Try reducing your reaction volume for the ligation. And consider using Klenow for the fill-in.
I would recommend that you optimize the vector:insert ratio. There's a few papers out there that addressed this problem theoretically. From my own expertise and others I have realized that a typical mistake is to start with too little insert.In case you can't get it to work you may want to try A/T-cloning or derivatization with terminal transferase.
A good idea is to ligate in the presence of the enzyme that you used to cut the vector. Providing the enzyme does'nt cut the insert. This way you don't have as much self-ligation of the vector. Good Luck