| Total RNA was isolated from Cheiranthus Cheiri L. leaflet. The cDNA library was constructed containing 6.2×105 recombinants using A. Triplex2TM as a vector.The library was screened with homeologous outer envelope membrane protein probe of garden pea. The results were follows:1. Construct the cDNA library of Cheiranthus Cheiri L. leafletCheiranthus Cheiri L. leaflet tissure were homogenized in a denaturing solution containing 4M guanidinium thiocyanate. The homogenate was mixed sequentially with 2M sodium acetate, pH4, phenol, and chlorlform/isoamyl alcohol. The resulting mixture was centrifuged, yielding an upper aqueous phase containing total RNA. In this single-step extraction the total RNA was separated from proteins and DNA that remain in the interphase and in the oraginc phase. Following isopropanol precipitation, the RNA pellet was redissolved sodium acetate and alcohol. Place the sample for 2h at -20℃. Centrifuged 20min at 10000g, 4℃, and discarded supernatant. Dried the RNA pellet in a vacuum for 5 to 15min,dissolved the RNA pellet in 300ul DEPC-treated water.Used the SMART which came from CLONTECH when conversion of mRNA into double-stranded DNA .With CDSII1/3' PCR Primer serving as primer and the 5' PCR Primer as a short, extend template at the 5'-end of the mRNA ,the full-length ss-cDNA was synthesized by MMLV reverse transcriptase.The full-length ds-cDNA was synthesized by LD-PCR .Becourse of the incorporation of asymmetrical sfiI restriction enzyme sites at the 5' and the 3' cDNA ends,the ds-cDNA was ligated ATriplEx2?vector after digestion with sfiI and size fractionation using a CHROMA SPIN-400 column .The vector was phosphatased to prevent ligation without a cDNA insert. After the ligation mixture packaged in vitro, the cDNA library of Cheiranthus Cheiri L. leaflet was contracted and stored at -70℃.2. To detemine the titer of the library and calculate the cloning efficiencyUsed E.coli XL-blue as the host for the cDNA library of Cheiranthus Cheiri L leaflet. In separate tubes, aliquots of eacli dilution were mixed with E.coil. Phages were allowed to absorb to the cells and the phage/cell mixture was then heated to 37℃, causing the phage to inject their DNA into the cells. Top agar was added to each tube and the mixture was poured onto rich plates, which were incubated at 37℃ until plaques appear. Each plaque contained phages derived from a single infecting phage. IPTG and X-gal were added in the plates to determine the fraction of the clone that represented recombinants. The cDNA library of CheiranthusCheiri L leaflet was constructed containing 6.2×105, the recombination frequency was 85%. To evaluate the library was to pick 7 single recombinant clons from the initial titer plate, and carried them througth the phage DNA miniprep. Resuspended the miniprep DNA in 25ul water and digested 10ul with sfil. Analyzed the digest by electrophoresis through a 1.1% agarose gel in TBE buffer. There were 4 recombinant clones to have inserts visible by ethidium fluorescence, with an average size of 500-750bp.In vitro packaged phages were unstable. If stored for more than a day or so, the liter of the storck would drop noticeably. Thus, the library must be amplified. The major concern with any amplification step was that each original recombinant be equally represented. Let bacteri containing the recombinant clones were grown on agar plate, washed off the plates, and stored in glycerol. The titer of amplifified library was 1.2× 109pfu/ml.3. The library was screened with homeologous outer envelope membrane protein probe of garden peaThe basic principle of screening cDNA library was bacteriophage plaques, containing relatively large amounts of insert cDNA that can be detected directly by hybridization. Bacteriophages were plate onto agar plates at high density so that as many as 1 million different plaques can be screened. The bacteriophage plaques were then transferred to nitrocellulose filters, denature, and baked. The filters were hybridiz...
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