I've been attempting to subclone an insert from one vector to another by PCR amplification with primers that incorporate XhoI/EcoRI sites at the ends of the insert and then digesting the insert and vector with both enzymes and performing a ligation. I've never had trouble with this kind of experiment before. I know that both enzymes are working, and I know that my ligase is working. I can see that the insert ligates to itself, and it appears that the vector is also ligating to itself as observed on a gel. I've tried 1:1, 1:3, 1:6 vector:insert ratios with no luck.
I have three suggestions. 1) dephosphorylate your vector to try and inhibit the vector religation and increase the probability that you will get an insert containing clone (even though for some reason the insert and vector are not ligating efficiently)
2) Run your cut insert and vector samples out on a gel, cut out the insert and vector bands, and then do an in-gel ligation (You basically just smash the excised bands from the gel up in ligation buffer and add ligase, incubate, and then you use a few ul of the liquid for transformation. Minimize the amount of time your bands are exposed to UV as much as possible.) I think that this helps in 2 ways, it will help separate unwanted pieces of DNA out of the ligation reaction, and by not carrying the DNA through a gel purification process it minimizes handling and may help keep the ends of your insert and vector intact.
3) I suppose it depends on what you are cloning, but it is possible that your insert is "toxic"? Since you have already grown it in another vector this is unlikely, but you should still keep it in mind.
Hope this helps you, good luck!
Becca
I agree with above suggestion, Dephosphorelation by Shrimp Alkaline Phosphatase (SAP) will prevent self ligation.
once it happened to me. you may try "Quick and Dirt Ligation" method. Just google it. But this is not for beginners. I got positive clones when i used quick and dirt method. Just try..... All the best.
Try ligate at temperature of 14-16 C overnight. It worked like magic to me in the past when I had unexplained difficulties.
Thanks so much for the suggestions. I have never heard of ligating in a gel. I will give it a try! I will also try dephosphorylation of the vector. I have tried ligating at different temperatures and for different periods of time.
@Becca: Regarding the In-gel ligation, TAE and EtBr do not affect the ligase activity? I did not understand this part. How does this process of ligation work? Please help me understand.
Regards
Shridhar.
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