Why does PCR fail after 5'-phosphorylation of primers using T4 PNK?

So, I've treated both of my primers separately with T4 PNK using folowing protocol: 1 µl of 100 µM primer + 1 µl of T4 ligase buffer + 1 µl of PNK (10000 U/ml) + 7 µl NF water in each of eight...

19 March 2022 1,394 6 View