Phosphorylating Adapters and Linkers
600 pMol of adapter/linkers
2ul kinase buffer
1ul 10mM ATP
2ul T4 PNK (Polynucleotidekinase)
20ul rxn vol
37C for 30 mins
Ligation of phosphorylated linkers to DNA
Ligations of blunt adaptors to blunt DNA should be driven by performing ligation in a small volume with a large excess of linkers.
7ul ligation buffer
H20 to 80ul
This however necessitates a large amount and vol of enzyme digestion following ligation to cut them back.