Efficient Creation of Sequencing Libraries from Blunt-Ended Restriction Enzyme Fragments

摘要

The insertion of blunt-ended DNA restriction enzyme fragments into similarly ended vectors is not as efficient as the ligation of fragments with cohesive termini (4). Apart from the considerations of ligase concentration and ATP inhibition, the self-ligation of the nonphosphatase-treated components of the reaction may give rise to large numbers of false-positive colonies. Here we describe an efficient method for the ligation of blunt-ended restriction fragments into a TA vector (pCR~(TM) II; Invitrogen, San Diego, CA, USA) for the production of sequencing templates.