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The Construction of cDNA Library of Human Placenta Tissue and Cloning of Human Growth Hormone

Author: LiuHongJun
Tutor: LiQingWang
School: Northwest University of Science and Technology
Course: Animal Genetic Breeding and Reproduction
Keywords: Placental tissue cDNA library construction Human Growth Hormone Clone RT-PCR
CLC: Q78
Type: Master's thesis
Year: 2007
Downloads: 156
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Abstract


cDNA library construction and screening is one of the important methods of cloning, it is found that the basic tools of new genes and gene function. From the cDNA library can be screened to the desired gene, and used directly for the expression of the gene. Classic cDNA library cloned fragment short exists disadvantages, while the full-length cDNA library can provide a complete mRNA of information, which is able to clone the full-length sequence of the target cDNA. This study human placental tissue as the material, the extracted total RNA using TRIzol reagent, to the LD-PCR (long-distance PCR)-based cDNA library was constructed, and the constructed library quality been identified, cloned human growth hormone cDNA. The long sequence provides a theoretical basis and technological foundation for the study of recombinant human growth hormone. To obtain concrete results. 1. Said precooled 100mg of tissue into the homogenizer, add 1ml denaturant TRIzol was homogenized; after centrifugation, the supernatant was added to 200μl chloroform extraction, the supernatant was added an equal volume of isopropanol precipitation; RNase-free water redissolving precipitated by adding 1/10 volume of sodium acetate and an equal volume of chloroform extraction repeated until the interface between the foam protein so far; adding an equal volume of precooled isopropanol precipitation with pre cold 70% ethanol and rinsed twice to dissolve the precipitate; with 30-50μlDEPC processing ddH2O extracting total RNA, and stored at -70 ° C. Total RNA was extracted with TRIzol reagent templates, F1, R2, F3, R4 for two pairs of specific primers, reverse transcriptase and polymerase ds-cDNA. Double-stranded cDNA digested by Sfi Ⅰ, T4 DNA ligase after the same digested JG45 plasmid vector into competent cells JM109, build human placenta cDNA library. 3 were analyzed by the capacity of the library, the recombination rate and the diversity of approximately 7.01 × l05 / μgds-cDNA, the recombination rate of 96% the capacity of the cDNA library, 100 randomly extracted cloned plasmid insertion sequence analysis, a total of 80 different cDNA sequences, and no repeated sequences. The results showed that the cDNA library constructed by this method has a fast, simple, efficient, library quality to meet the requirements can be used for large-scale gene analysis. 4 from human placenta separated extract total RNA, the method of using the RT-PCR (one step) and colony plasmid DNA extracted from the cDNA library as a template plasmid PCR amplified from the human growth hormone cDNA sequence, for found that its sequence is completely consistent.

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