FAQ: Will the PreCR Repair Mix blunt the ends of the DNA?

No. The polymerase present in PreCR, Bst polI, will fill-in 5’ overhangs, but will not remove 3’ overhangs. Furthermore, this polymerase will add a dA to the filled-in 5’ overhang. If blunt ended DNA fragments are desired after PreCR treatment use the following protocol.

Blunting Protocol. This protocol uses the optimal repair buffer (ThermoPol Buffer), but it is it is best to clean-up the DNA before performing the blunting reaction for two reasons. 1) the Bst polymerase, if active, will add a dA to the blunted DNA and thus compete with the polymerase chosen to perform the blunting reaction. 2) if the blunted DNA needs to be phosphorylated using T4 PNK then the (NH4)2SO4 present in the ThermoPol buffer must be removed because it is known to inhibit T4 PNK. The protocol starts with performing the DNA repair reaction as described in the PreCR Repair Mix datacard. After repair the DNA should be purified, ie spin column, ethanol precipitation. The purified DNA is subjected to blunting. For DNA blunting we recommend the Quick Blunting Kit (NEB#E1201). For blunting and ligation NEB has conveniently bundled the Quick Blunting and Quick Ligation kits (NEB#E0542).