why does ligase buffer precipitate? - is it ok just to vortex it?? (Jan/03/2007 )
the new kit (actually old but newly found 
) of ligation mix has this ligase buffer that i have to mix at least 30 minutes by vortex every time i want to use it. i still didnt check if the ligation is ok but i am wondering is it ok like that? what makes it precipitate? does it mean that it is very old?
Actually my old boss used to suggest us to thaw the buffer upto RT and then vortex and then use the buffer as it is and this should b fine.
I havent found any problem with if the precipiate is there or not. And yes, i have found precipitate even in a new buffer.
no idea what the percipitate in the ligase buffer is.
I hand warm my ligase to avoid percipitation. (Rather then incubate at 37 on a heating block.) In my experience, if the ligase is kept cool while defrosting, the percipitation problems goes away.
do you mean thaw on ice?
so you dont eliquot it?
i didnt find any difference have aliquotted the buffer compared to using it directly from the tube (provided one is careful not to contaminate it).
i thaw the buffer in my hands, somtimes in the 37 heat block.
do you mean thaw on ice?
so you dont eliquot it?
I do aliquote my ligase. I defrost the aliquote with my hands... I warm the ligase just enough for it to melt, it doesn't reach anywhere near body temperature.
sometimes like with Roche ligase buffer, it is written to avoid repeated thawing and freezing so I eliquoted it. But this new one, doesnt say anything, so im wondering 
.
.It's the ATP in the buffer that degrades upon repeated freezing and thawing. This is why many people aliquot it out -- if the ATP goes bad, your ligation will fail...
.It's the ATP in the buffer that degrades upon repeated freezing and thawing. This is why many people aliquot it out -- if the ATP goes bad, your ligation will fail...
that is why i have been wondering why it is ok for some companies not to eliquot it and others say it is not ok.
I think it's BSA which precipitates in ligase buffer when thawed, but this doesn't affect ligation efficiency.