precipitate in NEB T4 Ligase buffer?

K. Legate g9013118 at mcmail.cis.mcmaster.ca
Wed Feb 15 22:03:37 EST 1995


In article <D40F48.B83 at ncifcrf.gov>,
Paul N Hengen <pnh at fcsparc6.ncifcrf.gov> wrote:
>| The efficiency of my ligations goes way down after repeated freeze-thaw 
>| cycles of the buffer. Adding a small amount of ATP to the mix restores 
>| original efficiency.
>|
>...and how is the rATP stored? I keep mine at -20 C. If yours is also frozen
>at -20 C, then thawed before adding a small amount to the ligation buffer,
>wouldn't the rATP also degrade over several freeze-thaw cycles resulting in
>loss of ligation efficiency?
>
It's true the ATP itself would degrade over repeated freeze-thaws. But I 
don't really see a way out of that. We've taken to aliquoting our ligase 
buffer in small aliquots so there should still be enough ATP around until 
the tube has run out.
	The degradation of the ATP may also be a function of how it is 
frozen. We simply put the ligase buffer back in the -20C after use, but I 
tend to snap-freeze nucleotide mixes in liquid nitrogen before putting 
them back in our -70 C freezer.

kyle





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