DNA Ladder的制备

Preparation of DNA Ladder

背景:目前,制备DNA分子量标准的方法主要有2种,一种是用限制性内切酶消化某种DNA,另一种是利用PCR扩增,2种方法各有优缺点。在前期采用PCR技术在前期扩增100～500bp片段的基础上,实验室又成功扩增出600～1000bp片段,PCR产物经过纯化,混匀,制备的DNA Ladder,结果制备DNA Ladder的条带清晰,易于识别,可完全与公司商品化的DNA Ladder相比,完全可用于分子生物学实验。目的:利用PCR扩增技术制备DNA分子量标准参照物。方法:自行构建了一种特殊适宜扩增的质粒pUC-DNA,根据pUC-DNA的基因序列,利用primer5.0设计能特异扩增100～1000bp的PCR引物。PCR扩增出100～1000bp大小的DNA片段,在2%琼脂糖凝胶中电泳观察结果。用凝胶回收试剂盒回收目的PCR产物,测序结果与pUC-DNA上基因序列进行序列比对,Blast进行同源性分析。将PCR产物用酚/氯仿抽提,乙醇沉淀,按比例混匀,即可使用。结果与结论:利用PCR技术能够成功扩增出100～1000bp条带,片段大小与预期结果相符,片段序列与GenBank序列完全一致,利用回收片段制备的DNA Ladder条带清晰,可与同类产品相比。

BACKGROUND：DNA molecular weight standard（DNA Ladder） is one of necessary reagents in molecular biological laboratory.It can correctly measure the length of DNA fragments and serve as the DNA molecular weight standard.Now,there are two methods to prepare the DNA Ladder,one is PCR technique,the other is through DNA digestion by restriction enzyme.The two methods all have some merits and drawbacks.The first one can produce standard bands,but is difficult to obtain large bands.The second method is to prepare DNA Ladder restriction digestion of plasmid or phage DNA.In previous study,we have amplified 100-500 bp DNA fragments by using PCR technique,then purified the PCR product and prepared DNA Ladder.The bands of the prepared DNA Ladder were clear,higher definition and could be equal with that of commercial products.It could be used in the molecular biology experiments.OBJECTIVE：To prepare DNA molecular weight standard control.METHODS：A specific plasmid pUC-DNA for PCR amplification was constructed,according to the sequence of pUC-DNA.Primers were designed to amplify 100-1 000 bp DNA fragments followed by primer5.0.100-1 000 b p series DNA fragments were amplified and electrophoresised in 2% agarose gels.After identification by PCR recovery products and DNA sequencing,the DNA sequences were analyzed according to the pUC-DNA and the homology analysis was compared analyzed by blast.The PCR products were extracted by phenol/chloroform,precipitated with ethanol and mixed them proportionally.RESULTS AND CONCLUSION：100-1 000 bp DNA fragments were successfully amplified by PCR technique,and the size of the bands were the same as the expected.The purified bands were clear.The 100-1000 bp DNA ladder was prepared in our laboratory.The method is convenient and the size of the prepared DNA ladder is standard,it could be used in the molecular biology experiments.