Use EcoRI/KpnI, good combination? - (Dec/03/2005 )
Dear all,
I m going to use EcoRI and KpnI to clone my target gene. I would like to use Amersham One phor all buufer. Any Comment???
Thanks
siuchi
hi according to web catalog, the composition of ideal buffer for an eventual double digest is :
NEBuffer 1(yellow):
10 mM Bis Tris Propane-HCl, 10 mM MgCl2, 1 mM DTT (pH 7.0 at 25°C).
1X amersham buffer is : 10 mM Tris-acetate (pH 7.5), 10 mM magnesium acetate and 50 mM potassium acetate.
it seems that you have too much salts in this buffer and pH is not very appropriate.
The buffer 4 from neb is close o amersham : 20 mM Tris-acetate pH 7.9 @ 25°C, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol. And activity of KpnI in this buffer is 50%...
NEBuffer 1(yellow):
10 mM Bis Tris Propane-HCl, 10 mM MgCl2, 1 mM DTT (pH 7.0 at 25°C).
1X amersham buffer is : 10 mM Tris-acetate (pH 7.5), 10 mM magnesium acetate and 50 mM potassium acetate.
it seems that you have too much salts in this buffer and pH is not very appropriate.
The buffer 4 from neb is close o amersham : 20 mM Tris-acetate pH 7.9 @ 25°C, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM dithiothreitol. And activity of KpnI in this buffer is 50%...
Thanks very much I've done that double digest before with NEB Buffer 1 and BSA.
It works with high efficiency.
It receives my stamp of approval!
-Matt
hi,
According to my experiance from Roche company this combi (EcoRI and KpnI) is not 100% for any buffer but it works good using buffer A.
good luck,
Sada.