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cDNA Library Protocols OverviewKrizman Protocol 1 - Bidirectional cloning for both micro-bulk and microdissected tissues Oligo(dT)-primed double strand cDNA is produced from total RNA and adapter primers are added by blunt end ligation. The cDNA is subjected to 10-15 cycles of PCR amplification using adapter-specific primers and cloned into the pAMP10 vector by UDG-mediated cloning. Libraries are produced by transformation of DHB10 cells. Krizman Protocol 2 - Unidirectional cloning for both micro-bulk and microdissected tissues Oligo(dT)-based single strand cDNA is produced from total RNA and a primer of known sequence is added to the 5' end of all cDNA molecules by a reverse transcriptase- mediated strand switch event. Single stranded cDNA is used as template for 20-25 cycles of PCR amplification using primers specific for the strand switch primer sequence and an engineered tail sequence that is part of the oligo (dT)-based primer. The PCR product is subsequently cloned into the vector pAMP1 by UDG-mediated cloning. Libraries are produced by transformation of DHB10 cells. Various parameters were changed in the early development of the protocols using microdissected tissue to assess one or more technical aspects of library production. Since these research and development (R/D) libraries generated sequences which were submitted to dbEST they are included in the library browser for the sake of completeness. However, the gene profiles of these libraries are not necessarily reflective of the tissue type from which they were derived, and thus should not be utilized for determination of highly abundant genes, nor used with CGAP expression analysis tools such as xProfiler and DGED. Stratagene Non-Normalization Protocol This proprietary protocol has been used by Stratagene, Inc. in its CGAP library production efforts. Life Technologies Non-Normalization Protocol This proprietary protocol has been used by Life Technologies, Inc. in its CGAP library production efforts. These cDNA libraries are prepared using a patented RNase H deficient reverse transcriptase (see U.S.Patent Nos. 5,668,005, 5,405,776, and 5,244,797). The proprietary reverse transcriptase is intended to allow preparation of improved cDNA libraries having a higher percentage of full length cDNA molecules. Life Technologies Normalization Protocol This proprietary protocol has been used by Life Technologies, Inc. in its CGAP library production efforts. These cDNA libraries are prepared using a patented RNase H deficient reverse transcriptase (see U.S.Patent Nos. 5,668,005, 5,405,776, and 5,244,797). The proprietary reverse transcriptase is intended to allow preparation of improved cDNA libraries having a higher percentage of full length cDNA molecules. Soares Non-Normalization Protocol This protocol has been used by Dr. Bento Soares, Univ. of Iowa, in his cDNA library production efforts. This protocol has been used by Dr. Bento Soares, Univ. of Iowa, in his cDNA library production efforts. This protocol has been used by Dr. Bento Soares, Univ. of Iowa, in his cDNA library production efforts. |
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