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.

2ug DNA

10ul linker

7ul ligation buffer

2ul ligase

H20 to 80ul

o/n 16C

This however necessitates a large amount and vol of enzyme digestion following ligation to cut them back.