Problem with SmaI restriction digest

[Orangistae]

I'm trying to clone some genes into a vector by amplifying them and ligating the PCR products into a SmaI(=blunt-ended cutter)-digested plasmid, but I've been having trouble with it and I've narrowed down the problem to the digestion part. When I run some of the digest on a gel it looks fine, with a single bright band, in contrast to the double band of the undigested vector, but if I transform the digested, unligated vector into E. coli I get heaps of transformant colonies, which seems to indicate that there is a very high background of uncut vector. Can anyone help me work out what the problem is and what I can do about it?

I've tried the digest two times so far, using the same SmaI enzyme (Promega, and nowhere near its expiry date), buffer, and DNA sample [which was recently purified and is in EB buffer (i.e. 10mM Tris, pH 8.5) with 1mM EDTA]. The first time, I used 6ul DNA with 2ul each of enzyme and buffer in a 20ul reaction mix and incubated at 25 degrees (SmaI's optimal temp) overnight, before heat-inactivating, treating with TSAP, and transforming 0.125ul of the mixture into E. coli. The second time, I decreased the amount of DNA in the digest to 2ul, incubated the digest for 4hrs, and used 0.25ul for transformation (didn't bother with the TSAP). I got lots (>50) of colonies both times, although somewhat fewer the second time due to the smaller amount of DNA, and when I do the actual ligation and try the transformation with that, I don't get any more colonies compared to using the equivalent amount of 'digested' vector.

Any help or advice would be much appreciated.

[JackBean]

So, your problem is, that you get the same number of colonies in inligated and ligated reaction, right? 50 colonies is quite a lot, did you try to dilute it a little? Then you could see some difference

Did you use CIAP? Maybe if you used some exonuclease...

[Orangistae]

I did try reducing the amount of DNA in the digest to 2ul, and that did produce fewer colonies (from transformation of the digested, unligated vector) than before, but still far more than there should be for that amount of DNA, so I haven't tried ligation using that digest because I want to cut down the amount of background uncut vector first.

I did use TSAP to dephosphorylate the vector ends prior to ligation; that's basically the same thing as CIAP, right? Could you explain what you mean about the exonuclease?

[JackBean]

I meant to dilute bacteria after transformation (put it onto 2 plates instead of one;)

By exonuclease I meant, that it would degrade the unligated DNA, whereas circular should stay intact.

But IMHO the problem isn't in restriction, but in transformation. Did you try to put on plates only bacteria without DNA? What antibiotics do you use?

[Orangistae]

Ok, I haven't tried diluting the transformed cells before plating them, but there shouldn't be any transformants, or very few, for what is a fairly small amount of supposedly digested DNA, so I really shouldn't have to dilute them to get fewer colonies.

I still don't see how exonuclease would help; the problem seems to be that there is a lot of plasmid that is still uncut following digestion, so it wouldn't be susceptible to exonuclease.

I haven't tried bacteria with no DNA, but the number of colonies does seem to correlate with the amount of DNA used, so I think that indicates that the transformation part is ok. I'm using amp selection, with fairly fresh plates, and the amp stock itself isn't very old either.

[JackBean]

you wrote, that you have only one band on the gel, so I assumed, that it's cut completely.

And thus, the exonuclease would degrade the cut DNA

[Orangistae]

That's what I thought too, but surely the transformant colonies show that there is still uncut DNA present? You shouldn't be able to get any transformants using linearised DNA.