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5' end labeling of Oligonucleotides
Adhere to all safety procedures involved with handling radioisotopes!!!
|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
- Mix the first three reagents together, then add γ-dATP, and then finally add the PNK
- Note: Use filter tips when working with radioisotopes to prevent contaminating your pipetman.
- Incubate the mixture at RT for 30 min or more, behind proper shielding.
- Add 80 µL of TE¬8 to the Kinase reaction mixture.
- Purify the reaction mixture (100 µL) using the mini Quick spin Oligo Columns (Roche), stored in the 4˚C Fridge.
- Note: You can prep the column ~5min before the Kinase reaction is complete
- After purification check both the column and the flow-through for radioactivity, both should be very hot.
- 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.