Posted 12 August 2004 - 01:41 AM
my problem is, that Nar I does not want to cleave my DNA. Neither after 3h, nor 16h nor 24h of digestion at 37°C.
I read about using spermidine to improve cleavage activity of Nar I.
Has anybody tried yet? I`d like to know the concentration of spermidine that you are using and what kind of solvent are you using to dissolve it.
What about this "oligo-nucleotide"-stuff ? Some use oligo-nucleotides which contain a Nar I restriction site for activation. How big is " oligo-nucleotide" and what ´s the common concentration?
You see, not only cloning is a problem....
Looking forward to your replies!
Posted 12 August 2004 - 03:36 PM
this is just some stuff I founf in NEB catalog, so aaI apologise if you've already thought of it all:
1)What's your reaction pH- apparently at pH7.0 the digestion needs to be at 25C, not 37C
2)Some sites apparently are cut really slowly, so go with the longer incubation time
3)Is your DNA methylated by any chance- this enzyme is blocked by CpG methylation
I'd check your enzyme on some other DNA.
Posted 12 August 2004 - 11:47 PM
I have checked the NEB catalog as welll, but didn´t get any further.
CpG methylation can be excluded, as I´m not cutting genomic DNA.
The pH of 7 @ 25°C are in my opinion buffer specifications.
I even got that far to read the primary literature cited in the NEB!
But until now, Nar I still refuses to cleave!
Thanks a lot!
Posted 13 August 2004 - 03:26 AM
I'm sure you meant that, but just to exlcude everything. Not only genomic Dna can be methylated, but plasmid DNA also. And you sequenced your DNA to insure the site is really there? Somtimes synthetic oligos can be faulty - I once experiend a situation where one nucleotide forming the restriction site simply wasn't there in the original oligo - though ordered otherwise!
Otherwise i can just give the advice to change your supplier of enzyme. I once had a similiar problem with one enzyme, that refused to cut. I canged to the same enzyme from another supplier and - it worked as it should have done the whole time.....
beware I'm not saying NEB enzymes are bad - I never would. But sometimes one enzyme from another supplier maybe better than the other.