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PLoS One. 2015 Feb 3;10(2):e0116997. doi: 10.1371/journal.pone.0116997. eCollection 2015.

A colony multiplex quantitative PCR-Based 3S3DBC method and variations of it for screening DNA libraries.

Author information

1
Department of Biophysics, Kyoto University, Kyoto, Kyoto, Japan.
2
Comparative Genomics Laboratory, National Institute of Genetics, Mishima, Shizuoka, Japan.
3
School of Pharmacy, Fudan University, Shanghai, China.

Abstract

A DNA library is a collection of DNA fragments cloned into vectors and stored individually in host cells, and is a valuable resource for molecular cloning, gene physical mapping, and genome sequencing projects. To take the best advantage of a DNA library, a good screening method is needed. After describing pooling strategies and issues that should be considered in DNA library screening, here we report an efficient colony multiplex quantitative PCR-based 3-step, 3-dimension, and binary-code (3S3DBC) method we used to screen genes from a planarian genomic DNA fosmid library. This method requires only 3 rounds of PCR reactions and only around 6 hours to distinguish one or more desired clones from a large DNA library. According to the particular situations in different research labs, this method can be further modified and simplified to suit their requirements.

PMID:
25646755
PMCID:
PMC4315571
DOI:
10.1371/journal.pone.0116997
[Indexed for MEDLINE]
Free PMC Article

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