blunt end ligation problems - (Feb/17/2005 )

Hi! I am trying to make my plasmid shorter by using a blunt end enzyme to cut out a 3000 bp fragment and then let the rest part of the plasmid self ligated.
Basically, I did like follows:
1. Diagest the plasmid with Hinc II.
2. Gel purify the fragment I want.
3. Do ligation and transform into DH5a

However, I tried several times and just could not get any clone.

What is the problem? Is there anything wrong with my idea?

Thanks.

how short will the resulting plasmid be? how long are you ligating? I hope you are not cutting into a resistance or selection gene.

The resulting plasmid is around 6 kb. And I ligated for 16h-24h followed by the blunt end ligation protocol from invitrogen T4 ligase. I think the resistance gene should be intact after digestion. Thanks for your reply.

I dont see anything wrong with your ligation protocol. Try troubleshooting your transformation protocol.

Usually, when you buy competent cells, they come with a control plasmid. or you can use any other plasmid that you know works.

Yes. I do have a transformation control. They work normally. So it seems that it is not the problem of transformation.

Hmm. Try changing the water that you use. Get one fresh from the source.

It may sound silly and simple but sometimes it does the trick.

I may question the activity of the ligase, but I would try changing the water first.

If that doesn't work, then I am out of ideas, my friend.

The only time I ever got blunt end cloning to work was when I spiked my buffer with fresh ATP (and I mean FRESH). But then, I wasn't trying to do a unimolecular ligation, which should be easier.