Construction and characterization of a full–lengh cDNA library from non-fresh Giardia lamblia

Authors:
  • JJJie Jiang
    Jie Jiang
  • WZWen-Yu Zheng
    Wen-Yu Zheng
  • MLMing-Luan Li
    Ming-Luan Li
  • XTXi-Feng Tian
    Xi-Feng Tian
  • XFXian-Min Feng
    Xian-Min Feng
  • YWYue-Hua Wang
    Yue-Hua Wang
  • XJXiao-Hong Ju
    Xiao-Hong Ju
  • YKYue-Qiong Kong
    Yue-Qiong Kong
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Abstract
To construct rapidly a full-length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent. Total RNA of Giardia was extracted using Trizol reagent. A full-length cDNA library of G. lamblia trophozoites was constructed by a long-distance PCR (LD-PCR) method. The recombinant rate and the coverage rate of full-length clones of the library were evaluated. The inserted fragments were identified and sequenced by PCR amplification. The titer of cDNA library was 3.85 × 10(7) pfu/mL. The length of inserted fragments ranged from 0.4 to 2.5 kb, and the recombination efficiency accounted for 100% (20/20). The coverage rate of full-length clones is high (17/20). The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature. The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.

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