Partial digest to destroy an enzyme site - (Dec/05/2005 )

I have a plasmid that contains two BamHI sites. I want to destroy one of these so I can use the second BamHI site to do my next cloning step.

My method is:
1) Partial BamHI digest. (Choose the digest conditions that result in the half of the DNA being only cut once)
2) Klenow treatment to destroy the BamHI site
37 degrees for 30' then EDTA and heat inactivation
3) Gel purfiy the correct fragment (1x BamHI cut)
4) Ligate this vector (which now has blunt ends) back together
5) Transform and ID colonies to determine which BamHI was destroyed.
Results:
All the colonies still have two BamHI sites. My klenow step isn't blunting?? Any idea's?

Should I gel purify the band which has been only cut once (away from the 2x cut and uncut) and then klenow treat?

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This sounds like cloning from the 80's -- impressive when it works, but hardly necessary now. Here in the future, consider PCR mutagenesis of the BamHI site using the Stratagene Quikchange kit.

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Thanks for that.
I guess my excuse is that I'm a chld of the 80's...or maybe that my supervisor did mol biol in the 80's!!
Cheers.

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I am a man of the aughts...and I think Mung Bean Nuclease blunting and blunt ligation for the removal of restriction sites is way better/cheaper than mutagenic PCR. I've seen recent papers do this.

Just my opinion,

Matt

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Thanks Matt. (I also found a recent publication that used klenow to blunt and destroy RE sites!) My question now for you is why mung bean rather than klenow? Have you had experience using either of them to blunt?
Kate.

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Mung Bean blunts both 5' and 3' extensions while Large Klenow Fragment fills in 5' extensions but removes 3' extensions. If you are only dealing with 3' extensions, then it does not matter, but for blunting 5' extensions, you must use Mung Bean (unless you want to retain the site, of course).

I have not used Klenow, but I do use Mung Bean Nuclease in blunting sites for ligation of Gateway AttR casettes into non-Gateway vectors. We do high-throughput cloning and protein expression analysis, so Gateway cloning is essential.

I think you will find it quite difficult to find the conditions necessary that allows BamHI to cut only once. Are you sure there is not another Restriction site you can clone into? Is a blunt ligation impossible?

-Matt

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DOn't be worried, mentors with that kind of knowledge know more than nowdays kits, that is real molecular biology where you ahve to understand every step and why it's done, as oppose to kits where 99% of the students don't have a clue why they've used certain step as long as it yields results.

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Hey Matt,
My enzyme choice is very restricted!!
The other thing I tried was cutting my vector with BamHI (blunting that) and SacII then cloning in my fragment (that has the two BamHI sites) that had been cut with AgeI (and blunted) and SacII.
I tried that many times and had no luck.
Maybe I should go back to that and try mung bean instead of klenow?
Kate

Cheers Smoochiepie! That is very true! Anyone can do mol biol with a kit!
Kate

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If you're already doing a partial digest, you should produce four bands -- uncut, cut once, cut twice, and a band corresponding to whatever size fragment is liberated by the cut twice reaction.

Depending on how big a fragment exists between your two Bam HI sites, you should be able to separate the singly-cut population from the doubly-cut, uncut, and fragment bands. The singly-cut, linerized plasmid will exist as two spieces -- those that were cut at the site you want to clone into, and those that were cut at the other site.

If you can separate these bands, couldn't you recover the singly-cut population and use it as your vector in your cloning reaction, and find the right clone by screening the colonies?

I don't suppose the Bam HI site you don't want to use exists in some place required by the plasmid for replication or stability or something? Or perhaps in a selectable marker? Then your clones would be self-screening. Or if the Bam HI site you do want to use is in a lacZ-alpha gene for blue-white selection with X-Gal?

Anyway, if you control your partial digestion carefully (perhaps with a PCR machine, and done with various dilutions of Bam HI), and if you can separate the fragments on a gel, perhaps you could avoid trying to blunt end it and such...

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Of course!!!
I can partially digest my plasmid with BamHI and gel purify the 1x cut band then cut with SacII and gel purify the correct BamHI-SacII fragment (the 4 potential fragments are quite different in size) and then clone that into my BamHI-SacII cut (and already waiting) vector.
Thanks!

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