NdeI digestion - Share your experience (Mar/05/2010 )

Hello guys

I'm trying to clone a 3000 bp PCR product in a vector derived from pBR322. The vector is around 5000 bp. The site I'm looking is a NdeI site. I will have to check for correct orientation, but this comes later. I discussed a bit with the lab which sent me the plasmid and they pass always through a subcloning step of the PCR product, since it seems NdeI is giving trouble when in oligos. I'm not a big fan of subcloning steps since they give me another step to worry about and then troubleshoot if something is not working
So I added 8 bases in the 5' of my oligos containing the NdeI site + the extra A the Taq should add at the end. Based on NEB the enzyme cuts well with 7 bases after the site. I did the digestion of both plasmid and PCR product and I do see a very small amount of colonies. The ratio I used was 3:1 and 6:1 insert:oligo. I gel purified the insert (and as usual this is giving me less colonies).

So can you share your experience with NdeI. I used the Fast Digest from Fermentas, digested PCR and plasmid for 6 hrs and heat inactivated for 5' at 65 C. Gel purified the PCR, and purified the plasmid with Wizard PCR clean up kit. I cipped the vector for 15 mins and used a fast ligation kit (they stated 5 mins is enough) for 2 hrs.

I do not see why I should get not many colonies. The only thing I could think of is NdeI not working on my PCR products

Please share your experience with this enzyme!!

Cheers

You can test this by ligating the insert alone and heat killing the ligase, then running a gel. Do not do this with "quick" ligase, but with normal ligase. You should not be using quick ligase except for blunt ligations. Normal ligase is quick ligase for cohesive ends, and gives far fewer problems.

You should see multimers or smears of higher mw fragments in your ligated band (run an unligated sample as well).

To figure out what is happening, you need good controls. Do ligations with/without cip treatment. Do a transformation control (10 pg of pUC19). Do a vector only ligation and transformation.

We can only guess what is wrong from a very long list without some controls.

phage434 on Mar 5 2010, 06:28 AM said:

We've used Nde I extensively with no notable problems. But we go through the subcloning step because it actually saves time in the long run on average than direct cloning into the final vector.

Have you ligated this PCR product (3000 bp) successfully with other restriction sites?
It's pretty big and after checking the concentrations of PCR and vector, you can use
http://www.promega.com/biomath/calc06.htm
to calculate the molar ratio of plasmid to ligand and set up a series of ligations. I suggest
1:3, 1:1, 3:1.

Generally: we digest the ligand and vector at 37C for 30 - 60 minutes without Fermentas Fast Digest. The extended digest time may be causing problems.
Treat with CIP at 37C for 30 to 60 minutes
Ligate with Quick ligase for 5 to 10 minutes

Just some ideas, but controls are the best way to go as there's no real way to know what's going on without them.