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Xho1 doesn't digest pEGFP.N2 completely, why?


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6 replies to this topic

#1 Curtis

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Posted 27 June 2009 - 12:42 AM

my XhoI doesn't cut my pEGFP.N2 completely. I'm using Fermentas fastdigest and I have tried 3 different XhoI up to now because I thought maybe my own XhoI is spoiled.

surprisingly EcorI and NheI cut 100% of the plasmid and I can see single band on the gel, but for XhoI it's like only 60-70% of the plasmid is digested.

as a result my ligation didn't work well and I got no colony on my agar plate. why is that?

#2 HomeBrew

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Posted 27 June 2009 - 04:09 AM

Two things I can think of:

If you're recovering the plasmid from eukaryotic cells, XhoI is blocked by CpG methylation, whereas the others are only blocked by some combinations of CpG methylation.

Even by Fermentas' own data, XhoI is very sensitive to buffer conditions (see here), with cutting efficiencies dropping quite sharply in buffers other than their "red" or "yellow" buffer. NEB supplies this enzyme with BSA, and says that more enzyme or extended digestion might be needed for complete activity if it's used in suboptimal buffer conditions (see here and here).

I have always been skeptical of "fast" digests -- and it certainly hasn't saved you any time here. Get a "regular" enzyme, use the correct buffer, add more enzyme than you normally would, maybe add a little BSA, and allow to digest overnight. See if you get complete digestion then.

#3 Curtis

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Posted 27 June 2009 - 09:21 AM

thank you Homebrew,

I couldn't find any normal XhoI in the lab, that's why I did with fastdigest. I'm a little confused because other enzymes work fine.

thank you for the links also, they are very informative.

I'm recovering from TOP10.

#4 Curtis

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Posted 27 June 2009 - 11:05 PM

something crazy happened, :( :) ;

Yesterday after posting this thread I threw away my digested sample. today I searched in the garbage box and found the tube and ran it on the gel after almost 20 hours. XhoI has digested almost 95% of the plasmid.

#5 HomeBrew

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Posted 28 June 2009 - 05:10 AM

It's not really crazy. XhoI remains active after >8 hours at 37C (according to NEB), so it's not too much of a surprize that it continued to digest at room temperature. As I suggested above, increase your incubation time -- do an overnight incubation if it's convenient -- and you should be okay.

#6 Curtis

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Posted 28 June 2009 - 05:16 AM

overnight incubation at what temp is better? 37C or 4C?

I think the garbage box was room temperature :)

#7 HomeBrew

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Posted 28 June 2009 - 06:01 AM

I would use 37C. At 4C, the activity of the enzyme per unit of time is greatly decreased, thus you're giving up much of the benefit of extending the digestion time by doing it a 4C.




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