Customers frequently ask “Do I need a 5’ phosphate for ligations?”
Yes! All ligations require a 5’ phosphate, which is generated through restriction digestions or kinase treatment of your fragments.
Primers ordered from a supplier do not come with a 5’ phosphate unless you specify that modification is necessary. If your primers are not phosphorylated, you’ll need to phosphorylate your PCR-amplified fragments with T4 Polynucleotide Kinase prior to ligation.
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