6 replies to this topic

[geneman]

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.

[george@CASE]

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

[geneman]

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.

[george@CASE]

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.

[geneman]

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

[george@CASE]

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.

[Great White Northern]

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.