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  • FAQ: What are the overhangs on the DNA ladder fragments? Can I end-label them using the T4 polynucleotide kinase (PNK)? What about the Klenow fragment?

    All the fragments in the 1Kb, 100bp, 2-log, 50bp, Low Molecular Weight DNA Ladders and the PCR Marker have 5’ overhangs that can be end-labeled with the T4 PNK or filled in using the Klenow fragment.

    Labeling with PNK can be done by the standard protocol of removing the existing phosphate groups from the fragments ends using CIP or the Antarctic phosphatase, but we also had excellent results using the much simpler “exchange” reaction, which utilizes the ability of the kinase to remove existing phosphates from the DNA and replace them with labeled phosphates. The procedure that worked best for us is as followed:
        -1ul T4 PNK
        -1ul 32P ATP (3,000Ci/mmol, 5mCi/ml)
        -2ul 10x T4 PNK buffer
        -1or 2ul DNA ladder (1ug)

    Add distilled water to a reaction volume of 20ul. Reaction is 30 minutes at 37C. Run the samples for 50 to 60 minutes at 100V in TBE buffer in a 4-20% acrylamide gel (10cm x 10cm). A 20 minutes exposure gives very readable signals. The signal strength is about twice that signal when ADP is added to 100uM.