precipitate in NEB T4 Ligase buffer?

Zal Suldan Z-Suldan at ski.mskcc.org
Wed Feb 1 08:44:08 EST 1995


Just a quickie:

Does anyone know what the white precipitate is that sometimes forms in
NEB
T4 Ligase buffer?

It seems to redisolve after heating to 37 C and vigorous shaking, but I
am
wondering whether repeated heatings may degrade the ATP or anything
else
in the reaction buffer.

Cheers,  Johnny D

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++


Same thing starting happening to us about a year ago when we used to
use Promega ligase. We figured out, and tech services confirmed, that
it's most likely the DTT coming out of solution at -20C. What you might
do is get a fresh tube of buffer (or make up your own) and then aliquot
out small volumes, each good for only a few ligations. You might also
think about spiking the ligation reaction with rATP part way through
the incubation since the rATP degrades during the reaction anyway.

Zal



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