Blunting of DNA from Digested Plasmid DNA

Gregory W. Broussard

Revised: 2/27/08

(Please use, change and disseminate freely!)

• Digest plasmid DNA to recover insert and vector fragments.

Ex: 23µL diH2O
20µL DNA
5µL NEB2
1µL HindIII
1µL NdeI
50µL, 37°C, 4hrs.

• Heat inactivate Restriction Enzymes if possible.

Ex: 65°C for 20min.
Or, remove enzymes by other method.

• Add BSA (if not already added), and dNTPs (100µM).

Ex: Add 0.5 µL dNTPs (10mM) and 0.5 µL BSA (10mg/ml)

• Add 1µL T4 DNA Polymerase (or 1 unit per µg DNA). Incubate for 15 min. at 12°C. (NOT ANY LONGER)

• Stop the reaction by adding EDTA to a final concentration of 10mM and heating to 75°C for 20 min.

Ex: Add 1µL 0.5M EDTA to the above reaction.

• Gel purify the fragment of choice.

Caution: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3′ to 5′ exonuclease activity of the T4 DNA Polymerase enzyme!!!