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Eppendorf Perfectprep Gel Cleanup Kit

Eppendorf Perfectprep Gel Cleanup Kit

Blunt-end Cloning of DNA Isolated Using the Eppendorf Perfectprep Gel Cleanup Kit

George Halley
Eppendorf 5 Prime, Inc.
October 1, 2002 Introduction

Agarose gel electrophoresis is a method used to separate and analyze DNA fragments. This method can be taken a step further; the fragments can be purified from the gel for use in downstream applications. High quality DNA fragments of various sizes (70 bp 10 kb) can be purified from agarose gels using Eppendorfs Perfectprep Gel Cleanup Kit with recovery rates up to 96%. It is important that the isolated DNA is of high quality because most applications are sensitive to inhibitors. Cloning is a common downstream application in which a gel-purified DNA fragment may be used, and successful cloning is a good indication of the purity of the fragment. This application demonstrates the use of gel-purified DNA in a blunt-end ligation.

A 3.49 kb PCR fragment of Lambda DNA was digested with EcoRV to produce a 1.68 kb blunt-ended DNA fragment. The cloning vector, pUC19, was digested with SmaI resulting in a linearized vector with blunt ends. After digesting the DNA and separating it on an agarose gel, the Perfectprep Gel Cleanup Kit was used to purify the fragment and the linearized vector. These were subsequently used as insert and vector in blunt-end cloning reactions which were performed in triplicate. Due to salt sensitivity, blunt-end cloning is more difficult, and this scheme is a stringent test of the cloning capability of gel-purified DNA fragments.

pUC19 contains the
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