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?
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 follows:
-1 μl T4 PNK
-1 μl 32P ATP (3,000Ci/mmol, 5mCi/ml)
-2 μl 10x T4 PNK buffer
-1 or 2μl DNA ladder (1μg)
Add distilled water to 20 μl. Reaction is carried out at 37°C for 30 minutes. 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 100 μM.
