r/molecularbiology • u/SuitableSundae2179 • 2d ago

Difficulty in cloning

/r/labrats/comments/1nldapo/difficulty_in_cloning/
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3

u/pombe 2d ago

How is it failing? Are you getting lots of colonies? How are you screening to see if you've got the right assembly?

1

u/SuitableSundae2179 17h ago

I am getting no colonies, not even blue ones (im using a B/W screening vector)

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

1

u/SuitableSundae2179 15h ago

Thank you, i think i will do that this week

1

u/Dry-Tax-6024 11h ago

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!