Starting Material: 1-5 μg of blunt-ended DNA* (100-1000 bp).
*If starting with blunt-ended DNA that has been prepared by PCR or by end polishing, DNA must be purified to remove the blunting enzymes.
1. Mix the following components in a sterile microfuge tube:
Purified Blunt DNA: 1-5 μg
NEBuffer 2 (10X): 5 μl
dATP (10 mM): 0.5 μl (0.1 mM final)
Klenow Fragment (3´→ 5´ exo–): 3 μl
Sterile H2O: variable
Total volume: 50 μl
2. Incubate in a thermal cycler for 30 minutes at 37°C.
3. Purify DNA sample on one spin column.