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Supercoiling blocking restriction site?


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6 replies to this topic

#1 seanspotatobusiness

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Posted 16 August 2009 - 11:00 PM

I have a plasmid which I expected to be able to cut with a particular enzyme. It transpires that the enzyme does not cut (very much), even in its own buffer. The enzyme worked a few weeks ago on a different plasmid.

Our molecular cloning sage (every lab should have one :P) suggests that I heat the plasmid to 60 C for 5 minutes in order to relax it, since perhaps supercoiling is blocking the restriction site. Has anyone else encountered this? She is away until Tuesday, so I have a couple of questions to ask.Should I heat just the DNA and water or also the buffer? Wont the plasmid just coil back up as it comes down to the 37 C? If this doesn't work, would a nicking enzyme be more useful? I just want to get an insert out of the plasmid.

#2 Teagan27

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Posted 16 August 2009 - 11:31 PM

I have a plasmid which I expected to be able to cut with a particular enzyme. It transpires that the enzyme does not cut (very much), even in its own buffer. The enzyme worked a few weeks ago on a different plasmid.

Our molecular cloning sage (every lab should have one :P) suggests that I heat the plasmid to 60 C for 5 minutes in order to relax it, since perhaps supercoiling is blocking the restriction site. Has anyone else encountered this? She is away until Tuesday, so I have a couple of questions to ask.Should I heat just the DNA and water or also the buffer? Wont the plasmid just coil back up as it comes down to the 37 C? If this doesn't work, would a nicking enzyme be more useful? I just want to get an insert out of the plasmid.


Hi..

Which enzyme is it that you are using? Is this a newly constructed vector? If so were the RE recognition sites conserved during the cloning reaction? (yes i have done this before).

Might also be silly to ask.. but are you cooling the plasmid down completely before adding the enzyme? some enzymes are very sensitive to temperatures above the recommended incubation temp and denature very easily. I dont think supercoiling is a problem when doing restriction digests on a standard plasmid. How large is the plasmid?

Cheers

#3 seanspotatobusiness

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Posted 17 August 2009 - 12:24 AM

I have a plasmid which I expected to be able to cut with a particular enzyme. It transpires that the enzyme does not cut (very much), even in its own buffer. The enzyme worked a few weeks ago on a different plasmid.

Our molecular cloning sage (every lab should have one :P) suggests that I heat the plasmid to 60 C for 5 minutes in order to relax it, since perhaps supercoiling is blocking the restriction site. Has anyone else encountered this? She is away until Tuesday, so I have a couple of questions to ask.Should I heat just the DNA and water or also the buffer? Wont the plasmid just coil back up as it comes down to the 37 C? If this doesn't work, would a nicking enzyme be more useful? I just want to get an insert out of the plasmid.


Hi..

Which enzyme is it that you are using? Is this a newly constructed vector? If so were the RE recognition sites conserved during the cloning reaction? (yes i have done this before).

Might also be silly to ask.. but are you cooling the plasmid down completely before adding the enzyme? some enzymes are very sensitive to temperatures above the recommended incubation temp and denature very easily. I dont think supercoiling is a problem when doing restriction digests on a standard plasmid. How large is the plasmid?

Cheers



The enzyme is XmaI.

I don't know the history of the vector (it was contructed a long time ago (about a year, I think). I was told that if this doesn't work, it would be sequenced again, suggesting that it was sequenced at one point and found to be as expected.

I have not yet heated the plasmid to 60 C. That is my next experiment. I will certainly allow the mixture to cool before adding enzyme (although I expect the plasmid to re-coil upon cooling?).

The plasmid is about 4.2 kb.

#4 Qundo12

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Posted 17 August 2009 - 02:06 AM

I think diluting the plasmid is another choice, high DNA density can reduce the digestion efficiency.

Edited by Quasimondo, 17 August 2009 - 02:06 AM.


#5 Teagan27

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Posted 17 August 2009 - 02:22 AM

I have a plasmid which I expected to be able to cut with a particular enzyme. It transpires that the enzyme does not cut (very much), even in its own buffer. The enzyme worked a few weeks ago on a different plasmid.

Our molecular cloning sage (every lab should have one :D) suggests that I heat the plasmid to 60 C for 5 minutes in order to relax it, since perhaps supercoiling is blocking the restriction site. Has anyone else encountered this? She is away until Tuesday, so I have a couple of questions to ask.Should I heat just the DNA and water or also the buffer? Wont the plasmid just coil back up as it comes down to the 37 C? If this doesn't work, would a nicking enzyme be more useful? I just want to get an insert out of the plasmid.


Hi..

Which enzyme is it that you are using? Is this a newly constructed vector? If so were the RE recognition sites conserved during the cloning reaction? (yes i have done this before).

Might also be silly to ask.. but are you cooling the plasmid down completely before adding the enzyme? some enzymes are very sensitive to temperatures above the recommended incubation temp and denature very easily. I dont think supercoiling is a problem when doing restriction digests on a standard plasmid. How large is the plasmid?

Cheers



The enzyme is XmaI.

I don't know the history of the vector (it was contructed a long time ago (about a year, I think). I was told that if this doesn't work, it would be sequenced again, suggesting that it was sequenced at one point and found to be as expected.

I have not yet heated the plasmid to 60 C. That is my next experiment. I will certainly allow the mixture to cool before adding enzyme (although I expect the plasmid to re-coil upon cooling?).

The plasmid is about 4.2 kb.


ok.. again.. i dont think that supercoiling is much of a problem when digesting of plasmids. The only refernce to this i could find on the net said that you may need to just add a little more enzyme.

When you ran it on a gel was there some insert? and did the vector display the exact same pattern as the non-digested vector?

Some ideas:

*Try digesting with SmaI. This has the same recognition site. May confirm if there is a problem with the enzyme or not. Although this may not be helpful if you are wanting sticky ends for your cloning reaction it may confirm if you have a problem with the XmaI enzyme and that the recognition site is there?

*digest with a different enzyme if another restriction site is available. This will only linearize the plasmid, but it may help to confirm that the insert is actually in there if you compare the size to a marker and the base plasmid (also digested).

* Post your digestion protocol. Too little or too much enzyme may be a problem.

* test the enzyme again with the plasmid you digested a few months ago?

Good luck!

#6 klinmed

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Posted 17 August 2009 - 02:38 AM

I have a plasmid which I expected to be able to cut with a particular enzyme. It transpires that the enzyme does not cut (very much), even in its own buffer. The enzyme worked a few weeks ago on a different plasmid.

Our molecular cloning sage (every lab should have one :D) suggests that I heat the plasmid to 60 C for 5 minutes in order to relax it, since perhaps supercoiling is blocking the restriction site. Has anyone else encountered this? She is away until Tuesday, so I have a couple of questions to ask.Should I heat just the DNA and water or also the buffer? Wont the plasmid just coil back up as it comes down to the 37 C? If this doesn't work, would a nicking enzyme be more useful? I just want to get an insert out of the plasmid.

I have never had a problem, however empirically "older" plasmid preps seem to cut more easily.

Are you digesting overnight since NEB specify this with Xma I (they don't mention why!)

#7 seanspotatobusiness

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Posted 24 August 2009 - 11:10 AM

Just to let you know, I tried with the (neo)isoschizomers Cfr91 and SmaI and neither cut, so I figure the site isn't there. Fortunately, we found some other source of the fragment that I wanted, so I didn't get too bogged down.




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