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SacI enzyme trouble


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#1 lotus

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Posted 10 December 2009 - 01:46 PM

Hello, i am trying to clone a 1.5 kb insert into a 3.8 kb vector using NheI and SacI enzymes. I am using NheI-Hf and SacI from NEB. NEB recommends double digestion. I have tried this as well as sequential digest and it is notw orking. NEB also says sacI is salt-sensitive and DNA might have to be ethanol precipitated before cutting. is this necassry?
has anyone encountered problems with using sacI?

#2 HomeBrew

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Posted 10 December 2009 - 07:44 PM

I haven't had problems with SacI, but I have with NheI -- especially if you're trying to cut an NheI site engineered on to a PCR product. NheI doesn't cut well on the ends of PCR products -- see here. When we're forced to use it, we always have to go through an intermediate TA cloning vector first, then pop the insert out of this vector by digestion, clean up the fragment by gel purification, and then clone it into the ultimate vector.

#3 Vini

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Posted 10 December 2009 - 10:49 PM

Hello, i am trying to clone a 1.5 kb insert into a 3.8 kb vector using NheI and SacI enzymes. I am using NheI-Hf and SacI from NEB. NEB recommends double digestion. I have tried this as well as sequential digest and it is notw orking. NEB also says sacI is salt-sensitive and DNA might have to be ethanol precipitated before cutting. is this necassry?
has anyone encountered problems with using sacI?



Hi Lotus,
I hv used both the NEB enzymes (NheI and SacI) extensively for my cloning work n both work really well. regarding EtOH pptn of DNA b4 SacI digestion, I dont think its necessary coz I hv always been doing it in the normal way....n i believe that NEB actually recommends buffer 1 for digestion wid NheI n SacI (i ckhd the catalogue)....did u try out a single digestion wid each of the enzymes to linearize ur vector n to c that the vials that u r using are fine???

Best...




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