Restriction, Klenow Blunting, and Re-ligation all in one Tub

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Restriction, Klenow Blunting, and Re-ligation all in one Tub

Postby carmeyeii89 » Mar 15 2013 2:05 pm

Hi!

I’m trying to make a single HindIII site out of two sites: HindIII and XhoI in a plasmid, for which I need to cut with those two restriction enzymes, blunt the ends with Klenow and religate the plasmid so I’m eft with only the HindIII site.

After the digestion, can I add Klenow, and then ligase all in one tube, i.e., without wasting time on an extra gel extraction step?

I’ve read that Klenow can be added directly to the restiction digestion, but I’m curious as to if one can further re-ligate intramolecularly directly as well.

Thanks for your input!
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Re: Restriction, Klenow Blunting, and Re-ligation all in one

Postby mchlbrmn » Mar 15 2013 10:53 pm

Perhaps, but it may not be recommended. Klenow can get into trouble if left unsupervised. Usually it is just incubated 15 min, or so, at 20-25C, with dNTPs (35uM) and then stopped with EDTA and heat (10mM, 75C 20'). You might get away with Klenow end filling, chilling, and adding ligase, supplement with the necessary ATP, and incubating at 16C or 4C, but the reaction is usually stopped first because the klenow polymerase can start running backwards and chew up the ends if incubated too long or warm.
Hmmm, if you were going to do only a 10 or 15 minute room temp ligation perhaps you could get away with it. You'd need to stop the reaction after 15 min, or conceivably, with luck, you could pop it directly into competent bacteria. I'm not sure if the klenow polymerase would have any feelings about the ATP being present for the ligase. This would be a blunt ligation, usually requiring an hour or two, so it may not get very far in only 15 minutes, although it is only an easy recircularization, so you might get away with it. High concentration ligase should work better.
I'm forgetting that the Hindiii must be inactivated so it won't cut the newly
Better would be to, cut the DNA with H3 + XhoI, heat inactivate 65c 20', add dNTPs and klenow, incubate 15 min 25C, add 10mM EDTA, heat inactivate 75C 20', add 20mM MgCl2 to replace the Mg++ removed by the EDTA (I was forgetting that), add ATP and ligase, incuabte 2 hrs, transform.
Am I forgetting anything? I think that should work, as long as the ligase doesn't get finicky about all the stuff present (it can be finicky about contaminants). You might increase the volume at the ligation stage to encourage circularization (which is favored anyway).
How do you check the final plasmid produced to confirm it is not a dimer? ? Run some uncut next to the original plasmid uncut?
If you left out the HindIII, you'd turn the Xho site into a PvuI site. Then, there would be no need to inactivate after restriction digesting.
If I weren't already so long winded already, I might add that the purpose of the gel extraction is more to remove any traces of uncut, supercoiled plasmid. A quick spin column would be sufficient to purify. Gel purification prevents background sometimes, but I think often a thorough digest can be sufficient, with luck.
Oops.
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Re: Restriction, Klenow Blunting, and Re-ligation all in one

Postby mdfenko » Mar 18 2013 6:26 am

what about the hindiii-xhoi fragment?

if you leave it in solution then it is able to religate (blunt ended, as well). you'll need to remove it either before closing the plasmid or separate after (by gel or by colony picking after transformation).
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Re: Restriction, Klenow Blunting, and Re-ligation all in one

Postby mchlbrmn » Mar 18 2013 8:26 am

Getting one DNA fragment to circularize is far easier and more rapid than getting a second DNA piece to ligate into it and then circularize; that becomes a second order reaction.
If the removed bit ligates back in, I think it probably won't matter, as long as the Xho site is gone, and H3 site(s) remain. It sounds like this does not need to be in an exact frame.
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Re: Restriction, Klenow Blunting, and Re-ligation all in one

Postby relaxin » Mar 18 2013 9:22 am

As they say, "More haste, less speed." The ligase uses a different buffer (no 50 mM NaCl) and incubation temperature and time. Even you heat-kill the Kenow, the ligase will not work as efficiently.
By all means, gel purify the cut and filled-in vector.

One more thing, can you simple cut with XhoI alone, fill-in and religate? This will destroy XhoI.
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Re: Restriction, Klenow Blunting, and Re-ligation all in one

Postby mchlbrmn » Mar 18 2013 11:27 am

NEB does officially approve of using T4 ligase in any of their main rest. enz. buffers supplemented with ATP.
The question was about whether this short cut would work, so with that in mind I reviewed the short cuts that can work. It is true that ligase can be sensitive to contaminants, so it is safer to column purify the vector before ligase. Gel purifying at some point will help remove any trace of uncut vector, which tansforms more efficiently than your desired cut vector, so it is recommended.
I also mentioned that a single Xhoi digest can be used, buried in there, to remove the xho site and leave the H3.
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Re: Restriction, Klenow Blunting, and Re-ligation all in one

Postby relaxin » Mar 18 2013 12:07 pm

mchlbrmn wrote:I also mentioned that a single Xhoi digest can be used, buried in there, to remove the xho site and leave the H3.

Yes, you did. I often do not read your long post carefully. :D
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