Abstract:

Objective   To construct the cDNA library of Demodex for further studying its related genes. Methods   Mites were collected by natural settlement in double distilled water and total RNA was extracted from the mites with the TransZol Up kit. The first-strand cDNA was synthesized by reverse transcription and the secondstrand cDNA was synthesized and amplified by LD(long-distance) PCR. After size fractionation with a universal DNA purification kit, the cDNA fragments longer than 500bp were ligated to the pGM-T vector. The ligation mixture was transformed into the competent cells of E.coli TOP10 to complete the construction of the cDNA library for the mites. The content and recombination rate of the cDNA library were tested and the length of inserted fragments was analyzed by PCR. Results   The content of the cDNA library was estimated as 2.56×106 cfu with the recombination rate reaching 98%. The average length of the inserted cDNA fragment was longer than 1000bp. Conclusion   The results showed that the quality of the cDNA library is good enough to set the basis for further exploring the genes of Demodex.

Key words: Mites; Gene library; Polymerase chain reaction