[Construction of cDNA library of Eimeria tenella sporulated oocysts].
A lambda ZAP express cDNA library was constructed using mRNA from Eimeria tenella sporulated oocysts. Total RNA was isolated by the TRIzol from Eimeria tenella sporulated oocysts, mRNA was further purified through oligo(dT)-cellulose columns. The first-strand cDNA was synthesized by using MMLV reverse transcriptase with oligo(dT)18 primers containing Xho I restriction site. After the second strand cDNA replacement synthesized, the uneven termini of the double-stranded cDNA were filled in with cloned Pfu DNA polymerase and EcoR I adapters were ligated to the blunt ends. Then the double-strand cDNA was digested with Xho I restriction enzyme. The fragments of 0.5 kb-4 kb were collected by agarose gel fraction method. After ligation of the cDNA with the lambda ZAP Express vector, the cDNA library was packaged using Gigapack III Gold Packaging extract. According to the phage plaques bright selection, the cDNA library contained 6 x 10(6) clones and the titer of the amplified library was 1 x 10(11) pfu/mL. By using PCR identification, the cDNA library contained approximately 96% recombinant phages.
Han
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HY
Key Laboratory for Animal Parasitology of Ministry of Agricultry, Shanghai Institute of Animal Parasitology, CAAS, Shanghai 200232, China.
Huang
B
B
Zhao
Q P
QP
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D016786:Eimeria tenella / Q000235:genetics*; D005784:Gene Amplification; D015723:Gene Library; D017125:Genes, Protozoan; D009865:Oocytes; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D013170:Spores / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D012333:RNA, Messenger