Bonehead ligation driving me crazy

Tim Fitzwater tfitzwater at somalogic.com
Wed Mar 6 10:42:49 EST 2002


> > A quick note: according to NEB and I quote "less than 50% of AflII 
> > fragments ligate in a standard 20ul reaction containing 100-500 units 
> > of T4 DNA Ligase" 
> 
>Yes, I found this note. But how in the hell does one get 500 units of 
>ligase into a 20 microliter reaction, anyway? My ligase is "high 
>concentration" ligase, and it's only at 5 units/microliter. Also, 
>what is the temperature of a "standard" ligation reaction? Room 
>temperature? 12 degrees? (No one seems to do 12-degree overnight 
>ligations any more, but they were common about a decade ago.) The 
>relevance of this note is unclear to me. 

NEB sells ligase at 400 and 2,000 Units/uL.  One Weiss unit corresponds to
67 cohesive-end ligation units as defined by New England Biolabs, so the
concentrations are 6 and 29.85 Weiss Units/uL.  Since the NEB catalog is
referring to their own ligation unit definition, 500 NEB units equals 7.5
Weiss units/20 uL ligation reaction.  Having said that, historically some
lots of NEB enzymes have not met their catalog descriptions.  You should
check the data sheet that came with your lot of enzyme.  

You might also try Afl II's isoschizomer BspT I.  

Tim Fitzwater
Scientist
SomaLogic, Inc.
1775 38th Street
Boulder, CO 80301-2603

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