Phosphorylation (Kinase)

Vectors and inserts digested by restriction enzymes contain the necessary terminal modifications (5' phosphate and 3' hydroxyl), while fragments created by the Polymerase Chain Reaction (PCR) may not. Typical amplification by PCR does not use phosphorylated primers. In this case, the 5' ends of the amplicon are non-phosphorylated, and need to be treated by a kinase, such as T4 Polynucleotide Kinase, to introduce the 5' phosphate. Alternatively, primers for PCR can be ordered with 5' phosphate to avoid the need to separately phosphorylate the PCR product with a kinase.

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