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Restriction Digests of DNA Isolated with the Perfectprep ,,, Gel Cleanup Kit

Restriction Digests of DNA Isolated with the Perfectprep Gel Cleanup Kit

George Halley, Eppendorf 5 Prime, Inc. Introduction

Agarose gel electrophoresis is used extensively to separate and analyze DNA that is subsequently used for downstream applications. Eppendorfs Perfectprep Gel Cleanup kit is used to purify DNA fragments of various sizes (70 bp10 kb) from agarose gels with recovery rates of up to 96%. Since the majority of DNA purified from agarose is to be used in other applications, it is critical that the isolated DNA is of high quality. One downstream application in which the DNA may be used is a restriction enzyme digest. Restriction enzyme digests are sensitive to inhibitors and are therefore good indicators of possible contaminants in purified DNA.

The Perfectprep Gel Cleanup kit was used to purify a 3.49 kb PCR1 fragment amplified from bacteriophage Lambda. The purified DNA was subjected to restriction enzyme digestion using EcoRI, EcoRV and HindIII. The digests were done in duplicate, and for comparison, the PCR product was also purified using a competitors kit and then subjected to the same restriction enzyme digests. In the EcoRV and HindIII digests, the DNA purified with the Eppendorf kit was cut as well as or better than the DNA isolated using the competitors kit. In all cases using EcoRI, the DNA purified with the Eppendorf kit was digested more effectively than was the DNA isolated using the competitors kit. This indicates that DNA obtained from agarose gels using the Eppendorf Perfectprep Gel Cleanup kit can be used in restriction enzyme digests, and the performance in these dig
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