precipitate in NEB T4 Ligase buffer?
Zal Suldan
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
Randy Haun
>From: jp...@mole.bio.cam.ac.uk (John Dixon)
>Subject: precipitate in NEB T4 Ligase buffer?
>Date: Wed, 01 Feb 1995 11:14:38 +0000
>Just a quickie:
>
>Does anyone know what the white precipitate is that sometimes forms in NEB
>T4 Ligase buffer?
I think it's the DTT. . .
>
>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
>
>--
>John Dixon Lab 44 (223) 334131
>Wellcome/CRC Institute Fax 44 (223) 334134
>Dept Genetics
>Cambridge University
>United Kingdom e-m: jp...@mole.bio.cam.ac.uk
John Dixon
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
Paul N Hengen
| 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.
Could it be ficoll? Keeping rATP at 37 C should have no effect on stability,
else we human types would be in lots of trouble - losing all that energy we
work so hard to get (?).
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Colin Rasmussen
Hengen) wrote:
>
> Could it be ficoll? Keeping rATP at 37 C should have no effect on stability,
> else we human types would be in lots of trouble - losing all that energy we
> work so hard to get (?).
>
It's the DTT. However, repeated freeze-thawing of ATP does have effects,
at least according to one supplier of T4 ligase.
K. Legate
Colin Rasmussen <crasm...@anat.med.ualberta.ca> wrote:
>
>It's the DTT. However, repeated freeze-thawing of ATP does have effects,
>at least according to one supplier of T4 ligase.
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.
kyle
John Dixon
> Just a quickie:
>
> Does anyone know what the white precipitate is that sometimes forms in
> NEB
> T4 Ligase buffer?
I also sent a query to neb e-mail support. They replied:-
"It turns out that the ppt observed is BSA, which is added as a
stabilizing agent for T4 DNA Ligase. The occurance of this ppt has no
adverse effects regarding enzyme efficacy. While ATP and DTT are labile
components of the reaction buffer, our experience indicates that repeated
freeze/thaw cycles over a period of six months result in no reduction of
ligase activity. The enzyme
itself is stable for at least 12 months when stored at -20C."
YourName
jp...@mole.bio.cam.ac.uk says. e-m: jp...@mole.bio.cam.ac.uk
Hi,re.your query about Ligmix buffer.We seem to have the same
problem with the precipitate.Have you tried using One Phor All buffer
instead,you need to add 10mM ATP to the final mixture.Hope this works
better.
Paul Bundock and Emma. E-mail- bun...@rulsfb.LeidenUniv.nl
K. Legate
Paul N Hengen <p...@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.
>|
>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