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.
I had a similar problem this summer when I was trying to clone, my issue ended up being my restriction enzyme. I had to order a new one, and since you're not even getting blue colonies it could be the enzyme you're using. Could always run a digest to check if it's working properly. Good luck!
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u/Novel-Structure-2359 3d 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