To construct a directional cDNA library from Chinese giant salamander Andrias davidianus liver by SMART(switching mechanism at 5′ end of RNA transcript)technique, we purified the mRNA from Andrias davidianus liver an...To construct a directional cDNA library from Chinese giant salamander Andrias davidianus liver by SMART(switching mechanism at 5′ end of RNA transcript)technique, we purified the mRNA from Andrias davidianus liver and the first strand cDNA was synthesized through reverse transcription by using a modified oligo(dT)primer(contained sfi ⅠB site). We used the SMART oligonucleotide (contained sfi ⅠA site) as a template so that the first strand cDNA could be extended over the 5′ end of mRNA. The double strand cDNA was amplified by LD PCR (long distance PCR) with the above two primers and then digested by sfi Ⅰ (ⅠA and ⅠB) restriction enzyme. After cDNA fractionation through CHROMA SPIN column, the double strand cDNA was ligated into the sfi Ⅰ digested λtripIEx2 vector and then the recombinant DNA was packaged in vitro . The content of the unamplified Andrias davidianus liver cDNA library is 1 5×10 6 in which the percentage of recombinant clones is about 98 9%. The titer of the amplified cDNA library is 1 0×10 10 pfu/ml and the average exogenous inserts of the recombinants is 1 25 kb. These results show that the Andrias davidianus liver cDNA library has excellent quality.展开更多
文摘To construct a directional cDNA library from Chinese giant salamander Andrias davidianus liver by SMART(switching mechanism at 5′ end of RNA transcript)technique, we purified the mRNA from Andrias davidianus liver and the first strand cDNA was synthesized through reverse transcription by using a modified oligo(dT)primer(contained sfi ⅠB site). We used the SMART oligonucleotide (contained sfi ⅠA site) as a template so that the first strand cDNA could be extended over the 5′ end of mRNA. The double strand cDNA was amplified by LD PCR (long distance PCR) with the above two primers and then digested by sfi Ⅰ (ⅠA and ⅠB) restriction enzyme. After cDNA fractionation through CHROMA SPIN column, the double strand cDNA was ligated into the sfi Ⅰ digested λtripIEx2 vector and then the recombinant DNA was packaged in vitro . The content of the unamplified Andrias davidianus liver cDNA library is 1 5×10 6 in which the percentage of recombinant clones is about 98 9%. The titer of the amplified cDNA library is 1 0×10 10 pfu/ml and the average exogenous inserts of the recombinants is 1 25 kb. These results show that the Andrias davidianus liver cDNA library has excellent quality.
