hamel at ccu.umanitoba.ca
Tue Mar 2 18:12:09 EST 1993
In article <9303012249.AA11977 at pelican.dbe.csiro.au> adeno at dbe.csiro.au (both lab) writes:
>What is the best enzyme and the protocol for DNA dephosphorylation these days ?
>Thanks for your comments in anticipation.
Calf Intestinal Alkaline Phosphatase (CIAP) work VERY efficiently, one
MUST ensure it's removal after using it though:
use about 0.05 units per picogram 5' ends
incubate 37oC for about an hour in manufacturers recommended buffer
after incubation, add EDTA (disodium salt, pH 8.0) to 10 mM final
add SDS to 0.1% (w/v), then Proteinase K to around 10-50 ug/mL
incubate 50oC for around an hour
then using equal volume phenol/chloroform/n-butanol (50:49:1)
perform a couple of extractions (if your DNA is smaller than around 15
kbp, vortexing for 15 seconds is recommended, otherwise invert many times
gently if DNA is larger) ... finish with a couple chlorform/n-butanol
(49:1) extractions, then alcohol ppt'n.
I've stuck with Boehringer Mannheim's CIAP, though others are fine (I've
had same B.M. CIAP stock since 1984 aliquoted in -70oC in 50%
glycerol as well as aliquots in +4oC fridge since couple of years
ago and they've all worked nice.
Bacterial Alk Phos (BAP) are more difficult to remove. Thus I've kept awa from
Temp sensitive alk. phos. (from arctic/cold tolerant organisms) are more
expensive and tend to be somewhat less efficient, though they DO work if
one uses around 1 unit per picomole 5' ends. They are heat inactivated
(around 60oC for 10 min, then easily removed by single phenol/chloroform
then single chloroform ext'n). Over the years we've tried several temp.
sens. alk. phos. and haven't found one as good as CIAP.
Manitoba Vet. Virol.
email: hamel at ccu.umanitoba.ca
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