Construction of a cDNA Library from the Testis and Sequence Analysis of the Ubiquitin Gene from Rana nigromaculata
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Kunming Institute of Zoology, Chinese Academy of Sciences
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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 SfiI digestion and fractionation,
cDNA (>500 bp) was ligated to λ TriplEx2 vector and packaged
with GigapackR III Gold Packaging Extract. The titers of optimal
primary libraries were 2.0×106 pfu/mL and 2.4×106 pfu/mL and
the titers of the amplified libraries were 0.48×109 pfu/mL and
3.0×109 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
GenBank. 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.
Keywords
Rana nigromaculata; Total RNA; SMART; cDNA Library; Ubiqutin
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