Restriction, Klenow Blunting, and Re-ligation All in One Tube?
After digestion, can I add Klenow and then ligase all in one tube without wasting time on an extra gel extraction step?
Molecular Biology Forum brings bench scientists together in an online
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week’s highlighted question comes from the RNA Methods forum.
I’m trying to make a single HindIII site out of two sites: HindIII
and XhoI in a plasmid, for which I need to cut with those two restriction
enzymes, blunt the ends with Klenow and religate the plasmid so I’m left
with only the HindIII site. After the digestion, can I add Klenow, and then
ligase all in one tube without wasting time on an extra gel extraction step?
A: Perhaps, but it may not be recommended. Klenow can get into trouble
if left unsupervised...
A: What about the Hindiii-Xhoi fragment? If you leave it in solution
then it is able to religate...
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