digestion with two REs requiring different buffer - (Oct/06/2005 )
Thanks a lot , fred. this is very helpful.
forforfor
QUOTE (fred_33 @ Oct 7 2005, 12:48 PM)
hi 
NEB ensures that 100% is possible to get in 2h at recommended temperature. But sometimes, digesting overnight allows you to get complete digestion if your prep is not that clean. Sometimes it allows to save time.
But in this case you should take care of the star activity (degradation whithout any target sequence, neb web site gives some tips on enzymes, and you can access enzyme by enzyme, which one is suspected to have star activity).
if enzyme shows a star activity, i do my digestion in a PCR tube with a program :
optimal temp for 4h
hold 4°C
from neb web site :
for 1µg DNA, 5units (0.5 µl is enough) for 2h digestion.
good week end to you too.
fred
NEB ensures that 100% is possible to get in 2h at recommended temperature. But sometimes, digesting overnight allows you to get complete digestion if your prep is not that clean. Sometimes it allows to save time.
But in this case you should take care of the star activity (degradation whithout any target sequence, neb web site gives some tips on enzymes, and you can access enzyme by enzyme, which one is suspected to have star activity).
if enzyme shows a star activity, i do my digestion in a PCR tube with a program :
optimal temp for 4h
hold 4°C
from neb web site :
QUOTE
Unit Definition:
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.
for 1µg DNA, 5units (0.5 µl is enough) for 2h digestion.
good week end to you too.
fred
-forforfor-