ApaI / Not I double digestion problem - (May/24/2006 )

I have a weird situation on my hands with this double digest. I tried to cut 6 different genes which were all cloned into the same vector. I did a sequential digest with (first) NotI (1h 37°C 20x overdigest) and then with ApaI (30°C 1h 20x overdigest).
3 of the genes were cut out perfectly. But the other three gave problems...
I redid the experiment and again one of the enzymes had cut and the other one didn't. I cut the same plasmids with either NotI and ApaI and both were cut properly. It seems that the enzymes work fine alone but when performed after eachother (order doesn't matter, i tried that) the second one fails...
I tried to use NheI instead of NotI (i have that unique site as well) and again the same thing. However in combination with BamHI ApaI cuts fine and i have the correct bands.

Does anyone have an idea what is happening? Could it be a bad purity of DNA (ethanol?)? Or is this a nice thing that DNA sometimes does?

greetz

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the first digest works, second doesnot work. So the enzymes are working fine.

How do u stop the first digestion and the start the second digest? I think here lies the clue to this problem.

let us know.

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How close are the restriction sites to eachother? Is there a lot of intervening sequence?

I think your issue is with the manner in which you are stopping the reaction prior to the addition of the second RE.

-Matt

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Thanks for your replies.

I actually didn't stop the reaction. With the first 3 genes it worked fine without so i thought it was enough to put it on gel and then purify it from the gel.

The sites are about 2000bp apart.

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Have you check if the Apa and Not buffers are compatible one with the other? Because NEB buffer for Not is completely uncompatible with Apa enzyme...
In this case, you could cut with one enzyme, following by DNA precipitation (so you remove that enzyme buffer) and then add the second both buffer and enzyme...
Nevertheless, if there is a buffer problem, you could use a intermediate buffer, in which both enzymes cut (maybe at 50%)... and perform the digestion, maybe you´ll need more enzyme or more time (or both). But in this case, I have no idea if you will have problems because of non specific digestion...
Good luck

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i did that already. As you say buffers are not compatible at all. Every time i digested i did a gel + purification in between the two digests.

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Hey

Just wanna know if you every found a reason or solution to this problem with ApaI / NotI double digestion.

Kind regards,
Rasmus.

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