Bonehead ligation driving me crazy
Michael L. Sullivan
mlsulliv at facstaff.wisc.edu
Sun Mar 3 14:41:26 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.
Note, there are two common ways that units are defined for ligase. There
is a conversion factor between the two units. For ordinary cloning, I just
use 0.5 ul!
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