Three way ligation problem - (Sep/12/2012 )

Hi all,

My vector is about 7Kb. The cloning site is EcoRI-HindIII, but later I found there is another EcoRI site 1Kb downstream of the vector. Good thing there is a NheI site, 70bp upstream of EcoRI-HindIII site.

I've done the following,
1)Restriction digest the vector with NheI and HindIII, DNA gel purified and drop dialyzed in water.
2)PCR my insert (350bp), restriction digested with EcoRI and HindIII, gel purified and drop dialyzed.
3)PCR, restriction cut and gel purified the 70bp NheI and EcoRI fragment. This fragment is essential for expression.

The ideal ligated product is NheI-----------EcoRI------------HindIII.

Then I tried DNA ligation using Invitrogen T4 DNA ligase with vector (65fmol), insert (165fmol) and fragment (165fmol). But so far no colonies yet (DH5alpha chemical transformed, Invitrogen). Please let me know if anyone has any suggestion?

Thank you,

Wen

Make sure that the ligase buffer hasn't undergone more than a couple of freeze/thaw cycles - the ATP degrades. If it has undergone freeze/thaw, you can supplement with fresh ATP in the place of some of the water in the ligation reaction.

Check that the restriction digests are working individually.

Can you do the ligations sequentially? this will give you a much higher chance of it working.

For experience a simultaneous ligation of 2 inserts in a vector is difficult. Just some ideas:
- Why don't you try to mutate (a simple site-directed mutagenesis) your vector to remove the second EcoRI site? Or are all the 6 bases of the site really necessary for expression?
- Can you change your insert? I mean, can you put a HindIII site upstream and downstream your insert and do only a HindIII ligation in the vector (you would need to check the orientation of your insert)?
- As Bob1 said, you can try first a ligation EcoRI with fragment and insert and then a ligation NheI and HindIII with the vector

Thanks for the suggestion and ideas!

For Bob1: I will try the sequential ligation reaction. Correct me if I am wrong since I've never done sequential ligation. I shall first ligate the fragment and insert, gel purify product (or drop-dialyzed), and then ligate with the vector.

For metionina: My colleagues have tried site-directed mutagensis on this plasmid but failed. The EcoRI site appeared to be part of the product. HindIII ligation in the vector isn't an option since the goal is to replace the original vector insert with my insert in the EcoRI and HindIII region.

I was actually thinking more of ligating in one of the inserts, transforming, mini-prep, confirming presence of insert, then going to inserting the second bit.

Just to make sure: You have some 5' extra bases beyond your restriction sites on your pcr primers. You have purified your PCR reaction prior to cutting with the enzymes. Correct?

I would recommend electroporation. Your plasmid is larger than 7 kb.