Home Protocols A-Tailing with Klenow Fragment (3'→5' exo-)

A-Tailing with Klenow Fragment (3'→5' exo-)

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.