| To construct the cDNA library of Tetranychus urticae for further studying its related genes, total RNA was extracted of T.urticae by TRIzol and the construction of cDNA library was used by SMART cDNA library construction kit. A modified CDS III/3'PCR primer and the SMART IV Oligo primed the first-strand synthesis reaction and the second-strand cDNA was synthesized and amplified by LD (longdistance)PCR. Then the PCR products were purified with a universal DNA purification kit, the double-stranded cDNA were ligated to the pDNR-LIB vector. The ligation mixture was transformed into the E.coli competent cells to complete the construction of the cDNA library of T.urticae. The content and recombination rate of the cDNA library were tested and the length of inserted fragments was analyzed by PCR. The content of library was 3.54×106 CFU/mL, the recombinant efficiency was 95.8% and the average of insert size was between 0.3 kb and 2.0 kb.The results showed that this library was of high quality to set the basis for further exploring the genes of T. urticae. |
