Restriction digest of pQE plasmid - Double digestion problem (Feb/23/2007 )

Hi guys,

I think sometimes I do have a problem in digestion process. Always can't get a nice cut. Only sometimes I can get nicely cut plasmid. What are the problems? I tried different concentration of enzymes as well as plasmid.

The conditions are following:

BamHI 0.5ul (10 units)
Sal1 0.5ul (10 units)

Plasmid 5-7 ul (about 1-2 microgram)
BSA 0.2ul
Buffer BamHI 2
Water top up to 20ul

Incubated at 37 Celcius water bath for 4 hours.
Inactivated in 55 celcius for 5 minutes

End up smearing (the whole area instead of just the bottom part).

What do you think causing the smearing? My uncut looks ok.

Thank you.

smearing
that is not good.

Smearing indicates DNA fragmentation, and in the absence of genomic DNA most likely indicates DNA degradation, indicating presence of nucleases.
These nucleases probably reactivated when the restriction buffer was addded.

I would suggest a treatment with phenol chloroform, maybe 2 or 3 times.
Etanol percipiate and washed with 70% ethanol

Once cleaned up, try digesting again.

If the problem still occurs after the phenol treatment, the water (used in the restriction digest) is probably contaminated.

P.S: are you tips, eppendorf tubes autoclaved? The problem (though rare in my experience) maybe come form there.

I checked. Not the water nor the tips nor the eppendorf tubes. What else can it be? Do you think the amount of plasmid being digested is way too much or not enough enzymes were used?

Let me think.

If this is not a nuclease problem.. perhaps there is too much DNA in the digest. The amount of enzyme being use would not cause a problem. The volume of enzyme has not exceeded 5% (the problem comes from the glycerol used to keep then enzymes in)

How about trying this. Cut the same volume of DNA (I assume you need this much of DNA and can not do with less), 5ul... but digest it in a volume of 50ul. Digest for 2hrs. (or overnight if you are not in a hurry).

See what happens.

I have had similar problems with sal1 before, with certain plasmids. Is it a complete smear or some smear with DNA in the right size? IF you could cut out some DNA, try it for ligation and you might need to screen some extra colonies, but you can get it.

Good Luck !!!

not likely, the quantity of enzyme in use is small. Correct buffer at correct concentration is being employed.

I have never actually seen BamHI star activity before. This enzyme has yet to misbehave on me.

And speaking of misbehaving, i forgot my 'raving' about the evils of salI... So here it goes....
SalI is evil. It cuts plasmid DNA relatively poorly. And the DNA fragments from plasmid's cut by SalI ligate poorly in my hands.

Thanks for all the replies. I don't think it will be star activity effect. As mentioned by perneseblue, enzyme quantity is small. Plus it works when I am cutting my cloning vector. Just that it didnt work for my expression fusion vector.

I will try what perneseblue advices. Will update later this week.
Oh... I was just thinking. Can I just cut the plasmid with 1 enzyme first and then only I use the other enzyme? Single digestion at a time.

Oh forgot to ask, what is the minimum amount of DNA for digestion if I want to do gel extraction from there? Apparently, only about 80% of DNA is being recovered after gel extraction. I need it for ligation and I need it in high amount. If 2-3 microgram, is it ok? Will it able to be cut by enzymes? Thanks