Yu:PNK label

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5' end labeling of Oligonucleotides

 Adhere to all safety procedures involved with handling radioisotopes!!!

Reaction Mix

Reagent 1x Addition
10x PNK Buffer 2 μL
Oligonucleotide ≥100 pmoles (for tRNA)
milliQ H2O to 18 μL
Gamma dATP (3000mCi/mmole)(~3.33 nmol/μL) 1μL
Note: Try to keep the dATP/Oligo ratio at ≥ 5:1
  1. Mix the first three reagents together, then add γ-dATP, and then finally add the PNK
    1. Note: Use filter tips when working with radioisotopes to prevent contaminating your pipetman.
  2. Incubate the mixture at RT for 30 min or more, behind proper shielding.
  3. Add 80 µL of TE¬8 to the Kinase reaction mixture.
  4. Purify the reaction mixture (100 µL) using the mini Quick spin Oligo Columns (Roche), stored in the 4˚C Fridge.
    1. Note: You can prep the column ~5min before the Kinase reaction is complete
  5. After purification check both the column and the flow-through for radioactivity, both should be very hot.
  6. Sample is ready for hybridization.
To facilitate the kinase reaction the oligonucleotide should have a 5'-OH.  IDT's oligonucleotides already exist like this and do
not need to be dephosphorylated first.