Method for Phosphorylating the 5' ends of DNA using T4 polynucleotide kinase (PNK):
T4 DNA ligase requires a 5’ phosphate on one of the DNA molecules to be ligated in order to join DNA, for this reason it is often necessary to phosphorylate DNA molecule prior to adding it to ligation, for example when blunt cloning a PCR product.
DNA Phosphorylation Protocol:
Nuclease free water: 4.5 ul
PCR product or other DNA (cleaned-up): 4 ul (*if your PCR product has 5' recessed or blunt ends, heat it to 70 degrees C for 5 minutes and cool on ice before adding it to the reaction)
10 x T4 DNA Ligase buffer: 1 ul (* ligase buffer is used because it contains ATP)
T4 Polynucleotide kinase: 0.5 ul
- Incubate the kinase reaction(s) for 30 minutes at 37 degrees C.
- The phosphorylated PCR product can then be used directly in a ligation reaction without clean-up (for example when performing site-directed mutagenesis).
- If the PCR product is to be ligated into a de-phosphorylated vector, heat-inactivating the PNK enzyme may be a good idea to prevent it from phosphorylating the backbone vector and leading to high background. This is achieved by incubating the reaction(s) for 20 minutes at 65 degrees C.
Author: Dr Ryan Cawood