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Constructing Genomic Libraries Using the pGEM®-T Vector
By 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
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.
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