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Ligations are tricky, lots of things can go wrong. If you don't clean your PCR product and your vector the digested ends/primers/extra nucletides can mess up the ligation. Your ligase could have gone bad (it goes off much faster than other enzymes). You might need to add some ATP. Often the ratio of insert to plasmid needs to be changed. If you are cloning a particularly large fragment it can be especially difficult.

2007-01-23 01:48:39 · answer #1 · answered by floundering penguins 5 · 0 0

because of the many digested pcr product also contain complemantry base sequence present on ligand

2007-01-23 01:12:49 · answer #2 · answered by gelani v 1 · 0 0

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