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Constructing genomic libraries using the pGEM®-T Vector

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LabFact #5

The pGEM®-T Easy Vector System protocol uses the 2X Rapid Ligation Buffer. Use the appropriate volume when setting up the ligation reaction.
(from #TM042)

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Abstract

An efficient method for constructing a genomic DNA library is presented using a thymidine (T)-tailed cloning vector. The method is based on ultrasonic cleavage of genomic DNA, blunting of the fragment ends with mung bean nuclease and addition of a single 3´-adenine nucleotide with Taq DNA polymerase followed by ligation into the pGEM®-T Vector. The method is especially useful for genomic DNA that is poorly digested with restriction enzymes due to, for example, DNA methylation, polysaccharides or tightly bound proteins.

Promega Notes 73, 26.

Yoshikazu Kawata, M.S., Ajit-Kumar Thankappan, Ph.D., Shin-ichi Yano, M.S., and Hiroyuki Kojima, Ph.D.

Osaka National Research Institute, Agency of Industrial Science and Technology, Osaka, Japan Permanent address: Central Food Technological Research Institute, Mysore, India

Publication Date: 1999

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