Can I directly digest PCR products? - (Aug/17/2005 )

Hello,

I just added XbaI and XhoI restriction sites to a cDNA using primers (I added 6bp of extra sequence upstream of the restriction sites for the enzyme to "sit on"). Before attempting to clone this insert, however, I need to remove those 6 bases by digesting with XbaI and XhoI. Can I directly digest my PCR reaction, or do I have to PCI/ethanol precipitate it and then digest? My only concern is that the Hot Star Taq buffer may interfere with the digest.

Thanks,
Bob

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If it was me, I would purify. I would have put more than 6bp of up/downstream bases also... but hey it may work.

Let me know if the Xho/Xba ligation works... personal reasons you know.

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How many bases do you put upstream? I'm also doing XbaI/XhoI digests for cloning. The NEB site (which may or may not be that accurate) recommends 5 bases upstream of your restriction sites.

Also, do you gel purify after PCR, and then digest? Or do you PCI/ethanol precipitate and then digest? I've always wondered about this too since it seems like you'd achieve considerably better yields using the latter.

-Hank

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In previous lab, I always carried out PCR purification or gel extraction before I do anything to the PCR product.
But now in this lab, grant is limited, so I try without purifying it, and it works !!!

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The back of the NEB catalogue gives the activity of enzymes in a primer extension mix (PRC reaction).

XbaI = +++ (~100%)
XhoI = ++ (~50%)

I'd ethanol precip. before digestion. You may have to gel purify after digestion, especially if your primers possess the restriction sites - you don't want primer dimers competing with your insert during ligation.

Again, varies with enzyme. The NEB catalogue also has some info on this at the back (for some reason, the 2003 catalogue has more info than the current one).

XbaI has 94% or 99% cleavage efficiency 1 bp away from a cut by PinAI or AgeI respectively. The difference may be due to some effect of the different ssDNA overhangs left by these cuts.

XhoI has 97% cleavage efficiency 1 bp away from an EcoRI cut. (or 3 bp if you include the ss overhang.)

So you shouldn't have a problem there.

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I always purify PCR product by nucleotide removal kit or PCR clean up kit before performing double digestion for cloning purpose.

It's good to have purified templates.

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i dont think the pcr mix should interefere

i am sure if it is for enzymes like ecor1

i think u should try out no better way

prajwal

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I haven't looked at the NEB catalog in some time, but it appears as though it differs from the information on their website. The website is a bit misleading though as it states in fine print something along the lines of "when adding restriction sites to PCR primers add 4 bases to the listed number".

Anyways, I'm going to try to double-digest the PCR mix directly and see how it goes. Otherwise this is going to bug the hell out of me and I'll always be wondering.

Thanks,
Hank

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Adding the REs to the PCR mix will likely cut the DNA, since they are likely active in a PCR reaction buffer. Unfortunately, the PCR enzymes are also still active. Very likely the overhang created by cutting the PCR product will be filled in by the active Taq enzyme in the PCR mix. Since you are relying on the overhang for cloning, this is not good.

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Very good point phage!

Bob, if you directly digest following PCR, let us know how your cloning turns out. I think I'll stick to purifying before digestion.

-Hank

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