IN-VITRO HEADFUL PACKAGING SYSTEM FOR CLONING DNA FRAGMENTS AS LARGE AS 95 KB

摘要

This invention relates to an in vitro headfulpackaging process for cloning foreign DNA fragments as largeas 95 kb comprising:modifying vector DNA by inserting a stuffer fragment into ablunt end producing site which is proximal to a pac site;digesting the product of step (a) to produce two vector armseach of which contains (i) a blunt end, (ii) another end whichis compatible with the foreign DNA fragment which is to becloned, and (iii) a loxP site; ligating the foreign DNA to theproduct of step (b) without generating concatemers; reactingthe product of step (c) with pac cleavage proficient extractand head-tail proficient extract wherein the ratio of largeheads to small heads in the head-tail extract is at least 5:1;infecting a Cre+ bacterial strain with the product of step (d);and recovering the cloned DNA.