Digestion with EcoRI and NdeI - any tips? - (Jul/06/2012 )

I'm trying to digest both pET-24b and an insert inside Strataclone blunt PCR vector using EcoRI and NdeI, in an overnight double digestion for each of these plasmids.
I haven't been seeing any colonies after ligation and transformation. I'm trying to narrow down the options of what went wrong and would like to hear an opinion regarding the digestion step.

My mix is:
2 ug DNA
1 ul NdeI by NEB
1 ul EcoRI by NEB
2 ul EcoRI buffer (recommended by NEB for this double digestion)
DDW to a total of 20 ul
Overnight in 37 deg


Have someone had any experience in double digestion using these enzymes?
Would sequential digestion be better here? If so, should the DNA be purified after digestion with the first enzyme (NdeI probably)?
Do you think the glycerol amount could be too high (although it should be ok according to NEB)?

Do you see the linearised product when you run the digest on a gel?

In the pET 24-b reaction I can't distinguish between cut and uncut plasmid; the plasmid is 5.3 kb and even when running the gel for a long time, I can't see separate bands if they are indeed there. The EcoRI and NdeI sites are pretty close in the mcs of the plasmid so there is no additional small piece visible when working with 1% agarose gel.
In the Strataclone vector with my insert I also can't tell if NdeI is cutting properly since the insert contains on its edges NdeI and EcoRI sites, but the insert is also flanked by EcoRI sites of the vector itself..

Nebo on Sat Jul 7 10:25:12 2012 said:

You should be able to - unless your prep contains a lot of nicked or partially digested DNA from the extraction procedure.

Nebo on Sat Jul 7 10:25:12 2012 said:

You'll pretty much never see the small bit chopped out, it is too small for EtBr to be sensitive enough to see unless you are loading massive amounts. Try a single digest for each and see if they cut individually. If they do, you can re-try the double, but you may find it easier to do them sequentially, perhaps even doing a clean-up (EtOH works well) between them.

Nebo on Fri Jul 6 20:33:26 2012 said:

Hi,
Since you don't see any colonies, I would guess your digest worked pretty well. Because in case only one enzyme has cut proper, then you will definitely get colonies after transformation, since everything will easily re-ligate.
Therefore, check your ligation conditions (ligase, buffer) as well as your competent cells.
Furthermore, the glycerol amount is totally fine for this reaction; so don't have to worry about that. But if you wanna check your digest, then do 2 separate single digests (both in the EcoRI buffer) and run it on a gel for 30 min - both reactions must give you a single band (run the undigested sample as well to compare!) - as already mentioned by bob1.
Another critical point is the overnight digest ... if you have star activity of NdeI in the EcoRI buffer, your fragment might be randomly cut! Again, a quick run on a gel tells you the truth! ...
I never do digests for more than 3 hours (depending of course on the amount of DNA and setup) ... and the enzymes usually work pretty well.

Good luck,
Cheers, Daniel

Thanks for both comments, will certainly follow your advice.
BTW, NdeI doesn't have visible star activity overnight in EcoRI's buffer (no extra fragments I can see), but I recently learned NEB recommend to refrain from overnight digestion with NdeI (regardless of the buffer I suppose) due to removal of some additional nucleotides. Too bad it was in the bottom of NdeI's FAQ, while in its general description it says NdeI is ok for overnight digestion..