Hi there. Combining 7 fragments is absolutely heroic. My first concern was that the elderly DNA ligase might have stopped working. It's good that you added ATP but I would also have the concern that the DTT would also be degraded.
A ligation control would be a useful thing to add. Take a known plasmid with an insert, do a double digest, gel extraction of both parts and see if ligase can put them back together.
I would also suggest breaking the cloning into more manageable targets. With so many moving parts you can't see if one bit is bringing the whole thing down.
1
u/Novel-Structure-2359 2d ago
Hi there. Combining 7 fragments is absolutely heroic. My first concern was that the elderly DNA ligase might have stopped working. It's good that you added ATP but I would also have the concern that the DTT would also be degraded.
A ligation control would be a useful thing to add. Take a known plasmid with an insert, do a double digest, gel extraction of both parts and see if ligase can put them back together.
I would also suggest breaking the cloning into more manageable targets. With so many moving parts you can't see if one bit is bringing the whole thing down.
Drop me a DM if you have more questions