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Restriction, Klenow Blunting, and Re-ligation all in one Tube, no Gel Extraction?

Restriction, Klenow Blunting, and Re-ligation all in one Tube, no Gel Extraction? - Molecular Cloning Forum

Restriction, Klenow Blunting, and Re-ligation all in one Tube, no Gel Extraction? - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 03-15-2013, 07:09 PM
Pipette Filler
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Default Restriction, Klenow Blunting, and Re-ligation all in one Tube, no Gel Extraction?



Hi!

Iím 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 Iím eft with only the HindIII site.

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

Iíve read that Klenow can be added directly to the restiction digestion, but Iím curious as to if one can further re-ligate intramolecularly directly as well.

Thanks for your input!
Carmen
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