Kpn1 restriction enzyme - (Mar/06/2007 )
QUOTE (Madrius @ Sep 25 2008, 11:51 PM)
I've found that Kpn1 from NEB often loses its activity, and rather quickly.. never knew why!
We have had similar issues with some NEB enzymes. Then, yesterday, I used a brand new tube, and it struck me how much DTT I could smell in the 10x buffer. No idea, but does anyone think the reducing power of the buffer might be effecting the activity of the enzyme? That is, if I use an old tube of buffer, is a lower level of reduced DTT going to affect my digestion?
-swanny-
QUOTE (swanny @ Sep 25 2008, 05:02 PM)
QUOTE (Madrius @ Sep 25 2008, 11:51 PM)
I've found that Kpn1 from NEB often loses its activity, and rather quickly.. never knew why!
We have had similar issues with some NEB enzymes. Then, yesterday, I used a brand new tube, and it struck me how much DTT I could smell in the 10x buffer. No idea, but does anyone think the reducing power of the buffer might be effecting the activity of the enzyme? That is, if I use an old tube of buffer, is a lower level of reduced DTT going to affect my digestion?
if i m not mistaken Kpn1 is a low salt baby so you might have to sequential digestion . as far as i am concerned getting the wrong band could be due to wrong ionic strength but then again , Kpn1 can't function at high salt conc. but Kpn1 is good enzyme because it can cleave close to end with high efficiency
-Hanming86-
I've heard that DTT is quite fragile to freeze and thaw. So swanny maybe right. But hanming86's comments make sence too.
-Madrius-