SapI enzyme - (Nov/19/2008 )
So I have been trying to cut the pGFPmut3.1 vector with the enzymes Sap I and PstI. PstI seems to digest okay, but I've been getting incomplete digestion with SapI. Even overnight is not so great. Anyone had any experience using this enzyme?
Seriously...NO ONE??
Hello Xiheartmicrobesx,
SapI is a difficult enzyme...
For example you have to mix it prior pipetting because it has the tendency to sink down in the tube.
On the other hand extended digestion won't help... SapI is not stable in reaction. So maybe your aliqout is even too old for usage...?
The other important thing is that SapI is only 50-75% active at 25°C.
I would recommend you to use BspQI instead. It recognizes the same sequence and does the same cut. But be careful: You have to incubate at 50°C, there is only 10% activity at 37°C.
Good luck!
Chakchel
SapI is a difficult enzyme...
For example you have to mix it prior pipetting because it has the tendency to sink down in the tube.
On the other hand extended digestion won't help... SapI is not stable in reaction. So maybe your aliqout is even too old for usage...?
The other important thing is that SapI is only 50-75% active at 25°C.
I would recommend you to use BspQI instead. It recognizes the same sequence and does the same cut. But be careful: You have to incubate at 50°C, there is only 10% activity at 37°C.
Good luck!
Chakchel
I know. I read the NEB website. I just bought the enzyme though. I also digest at 37 degrees always. Thanks.