载体浓度和感受态对大分子载体转化效率的影响

Effects of Vector Density and Competent Cells on the Large Vector Transformation Efficiency

[目的]研究载体量和感受态对阳性克隆率的影响。[方法]利用引物拼接PCR技术,自行设计26条引物合成一个835 bp的目的基因,将该基因与约20 kb的大分子载体连接构成重组质粒。在1 500 ng目的基因底物量的基础上,设置50、100、150、200、250、300 ng 6个梯度载体量,得到的重组反应产物再转化入Top10F'、DH5α、Stbl3、Epi400、JM108、SCSI 6种不同的感受态中,组成36个试验组合。[结果]不同载体量阳性克隆率由大到小依次为200、250、300、150、100、50 ng,200 ng的阳性克隆率最高可达75%,平均达28.5%。不同感受态细胞阳性克隆率由大到小依次为Stbl3、Top10F'、DH5α、JM108、Epi400、SCSI,Stbl3在任何载体浓度下均高于其他感受态,平均阳性克隆率为42.4%。[结论]载体量和感受态均明显影响阳性克隆率,最佳组合为200 ng的载体分子量配合Stbl3感受态,阳性克隆率可达75%。

Objective] To research the effects of vector quantity and competence on the positive cloning rate.[Method] With a known gene se-quence but in the absence of DNA template, we artificially designed 26 primers to synthesize a target gene of 835 bp in vitro using overlapping PCR technique.The whole experiment design with two factors and six levels （36 combinations） was applied to study the effects of the vector den-sity and competent cells on the large vector transformation efficiency.Based on the 1 500 ng target gene, the vector density grades were designed （50, 100, 150, 200, 250,300 ng）, and then the recombinant plasmids were transformed into Top10F’, DH5α, Stbl3, Epi400, JM108, SCSI. [Result] The positive cloning rates of different vector amounts from big to small were in the order of 200, 250, 300, 150, 100 and 50 ng.The maximum positive cloning rate of 200 ng reached 75%;and the average value was 28.5%.The positive cloning rates of different competent cells from big to small were in the order of stbl3, Top10F′, DH5α, JM108, Epi400 and SCSI.Stbl3 was higher than other competent cells under any vector density.And its average positive cloning rate was 42.4%.[Conclusion] Both the vector density and competent cells have significant effects on the large vector transformation efficiency.The optimal combination is C4 with 200 ng vector density and Stbl3, the positive cloning rate of which could reach 75%.