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  • 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.