论文部分内容阅读
目的优化并验证改良菌落PCR程序的有效性。方法经常规PCR扩增DHBV编码核衣壳蛋白与DNA多聚酶重叠区的部分核苷酸片段(DHBc-dp),纯化特异性扩增产物并连入pMD 18-T载体,转化DH5α感受态细胞后涂布于Amp加倍的LB平板,记为亲代样品(PS),37℃培养12h,室温放置4h～8h。无菌操作挑取细菌转入优化的PCR反应液扩增、鉴定;余菌体室温放置完成二次生长。PS剩余PCR鉴定产物继代转化并涂布LB平板,记为继1代样品(F1S),同样进行菌落PCR鉴定。选取PS和F1S中PCR强阳性样品进行扩增及菌液PCR、质粒PCR、质粒酶切和测序鉴定。结果挑菌后的菌落二次生长成面积扩大的菌苔;优化菌落PCR反应体系保持、甚或提高了原有方法的高度特应性和稳定性;携外源目的片段的重组质粒在菌落PCR反应过程中能保持结构完整,并能在继代转化宿主细胞中忠实复制与扩增。结论改进的菌落PCR程序,继承了既有菌落PCR程序鉴定重组克隆的特异性和高效性,并能有效预防阳性转化子的丢失;同时,在增加优势抗性菌落生长量的前提下有效地抑制了卫星菌落的生长。
Objective To optimize and validate the effectiveness of the modified colony PCR program. Methods A partial nucleotide sequence (DHBc-dp) of DHBV-encoding nucleocapsid protein and DNA polymerase was amplified by conventional PCR. The specific amplification product was purified and inserted into pMD 18-T vector and transformed into DH5α competent cells Coated on Amp LB double plate, denoted as parental sample (PS), 37 ℃ for 12h, room temperature for 4h ~ 8h. Aseptically pick the bacteria into optimized PCR reaction solution amplification, identification; the remaining cells were placed at room temperature to complete the secondary growth. The PCR products of remaining PCR were subcultured and LB plates were plated, which were labeled as F1S (1st generation) and identified by colony PCR. PCR strong positive samples from PS and F1S were selected for amplification and bacterial PCR, plasmid PCR, plasmid digestion and sequencing. The results showed that the colony grown after pick-to-grow was secondary grown into an enlarged area of the lawn; the optimized colony PCR reaction system maintained, or even increased the high degree of atopy and stability of the original method; the recombinant plasmid carrying the exogenous target fragment was used in a colony PCR reaction The process maintains structural integrity and faithfully replicates and amplifies in the progeny transformed host cells. Conclusion The improved colony PCR program inherits the specificity and efficiency of the established colony PCR program to identify recombinant clones and can effectively prevent the loss of positive transformants. At the same time, it effectively inhibits the growth of dominant colonies The growth of satellite colonies.