Quick Tips - Do I need a 5' phosphate for ligation?

This video provides tips for phosphorylating fragments prior to performing a ligation reaction.


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.

Remember, if you have any questions you can contact us at info@neb.com.

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