A full-length cDNA library from the testis of dark-spotted frogs （ Rana nigromaculata ） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the...A full-length cDNA library from the testis of dark-spotted frogs （ Rana nigromaculata ） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the testis and reverse transcripted into full-length cDNA using PowerScript reverse transcriptase. The first-strand cDNA was amplified using long-distance PCR （LD-PCR）. After Sfi Ⅰ digestion and fractionation, cDNA （ 〉 500 bp） was ligated to λ TriplEx2 vector and packaged with GigapackⅢ Gold Packaging Extract. The titers of optimal primary libraries were 2.0×10^6 pfu/mL and 2.4 × 10^6 pfu/mL and the tlters of the amplified libraries were 0.48 × 10^9 pfu/mL and 3.0 × 10^9 pfu/ mL, respectively. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. The libraries were converted into pTriplEx2 plasmids in E. coli BM 25.8 strain. The insert sizes were measured by PCR which showed most fragments were over 500 bp and the average length was 1.0 kb approximately. A positive clone of 1 171 bp was sequenced and named RnUb based on sequence similarity with the known ubiquitin genes in Gen- Bank. This sequence was a full-length cDNA with complete coding sequences, which indicated that the library built a base for screening the full-length cDNA. These data showed that this library attained to the requirements of a standard cDNA library. This library provided a useful resource for the functional genomic research of Rana nigromaculata.展开更多
基金The National Natural Science Foundation of China(30640048)the Natural Science Foundation in Anhui Province(01043202)
文摘A full-length cDNA library from the testis of dark-spotted frogs （ Rana nigromaculata ） was constructed with the SMART （switching mechanism at 5＇ end of RNA transcript） technique. Total RNA was extracted from the testis and reverse transcripted into full-length cDNA using PowerScript reverse transcriptase. The first-strand cDNA was amplified using long-distance PCR （LD-PCR）. After Sfi Ⅰ digestion and fractionation, cDNA （ 〉 500 bp） was ligated to λ TriplEx2 vector and packaged with GigapackⅢ Gold Packaging Extract. The titers of optimal primary libraries were 2.0×10^6 pfu/mL and 2.4 × 10^6 pfu/mL and the tlters of the amplified libraries were 0.48 × 10^9 pfu/mL and 3.0 × 10^9 pfu/ mL, respectively. The percentages of recombinant clones of primary libraries and amplified libraries were all over 90%. The libraries were converted into pTriplEx2 plasmids in E. coli BM 25.8 strain. The insert sizes were measured by PCR which showed most fragments were over 500 bp and the average length was 1.0 kb approximately. A positive clone of 1 171 bp was sequenced and named RnUb based on sequence similarity with the known ubiquitin genes in Gen- Bank. This sequence was a full-length cDNA with complete coding sequences, which indicated that the library built a base for screening the full-length cDNA. These data showed that this library attained to the requirements of a standard cDNA library. This library provided a useful resource for the functional genomic research of Rana nigromaculata.
