I have tried In-fusion cloning few times and it used to work in my hand. Suddenly, after few months when I again wanted to do some more cloning, it is not working as in I am not getting any clones in the test plates.
I have used gel purified vector (fully double digested as observed in gel) and gel purified PCR products. The homology overlap of the PCR products were 15 bp.
I have used 50ng of vector and the Vector : Insert molar ratio was 1:4. The In-Fusion reaction incubation was done in a water bath set at 50C for 1 hr. It would be of help if I can get any advice regarding this.
If you still have material (vector + insert) from the previous cloning that worked, you could use it to test whether the InFusion mix is still active.
Thanks Pierre. I do not have the same batch of restriction digested vector. I am using a new batch.
It is most likely that your in-fusion rxn mix is inactive. Several freeze/thaw cycles will reduce enzyme activity. Did you make small aliquots for storage to prevent freeze/thaw cycles? How are you storing your mix?
I think in-fusion is the same as the abm pro ligatio-free kit E087. You can try their cloning kit, I have very good results with it and its cheaper than most kits.
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