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Restriction, Klenow Blunting, and Re-ligation All in One Tube?

03/18/2013
BioTechniques staff

After digestion, can I add Klenow and then ligase all in one tube without wasting time on an extra gel extraction step?


The BioTechniques Molecular Biology Forum brings bench scientists together in an online community to ask for and share advice regarding laboratory problems. This week’s highlighted question comes from the RNA Methods forum.

The BioTechniques Molecular Biology Forum brings bench scientists together in an online community to ask for and share advice regarding laboratory problems.




Q: 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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Keywords:  troubleshooting forum