如何准备转染所需的质粒DNA

细胞转染效率受到诸多因素的影响，除了细胞、培养基和载体等影响因素外，另外一项非常重要的因素便是DNA的质量。为了比较不同厂家的转染效率，我们分别从三家不同的供应商购买了质粒制备试剂盒来制备pEGFP-N3质粒DNA，并通过不同的方法将制备的pEGFP-N3质粒转运至NIH-3T3细胞。pEGFP-N3质粒分别通过Endofree Maxi Piasmid Kit(Qiagen),PerfectPrep Endofree Piasmid Maxi Kit(5 PRIME,inc)及NucleoBond DNA Maxi Kit(MACHEREY-NAGEL GmbH&Co.KG)制备，我们测定了230,260,280及320nm的吸光值检测DNA的质量。通过上述三种方法制备的DNA质量如下：MACHEREY-NAGEL＞5Prime＞Qiagen。pEGFP-N3由PolyJetDNA转染试剂运至NIH-3T3细胞。

细胞转染效率的高低与DNA的质量相符，由MACHEREY-NAGEL纯化DNA对应的细胞转染效率最高，而由Qiagen纯化DNA对应的转染效率最低。因此，MACHEREY-NAGEL GmbH & Co.KG的质粒制备试剂盒被认为是制备转染级别DNA的最佳选择。

How to prepare transfection grade plasmid DNATransfection efficiencies are affected by a variety of different parameters.  Besides factor such as cell culture, medium and vectors, one of most critical parameters is DNA quality.  We prepared pEGF-N3 plasmid DNA by large plasmid preparation kits from three different vendors and tested transfection efficiencies by delivering pEGFP-N3 DNAs prepared by different methods to NIH-3T3 cells.  We prepared pEGFP-N3 plasmid with Endofree Maxi Plasmid Kit (Qiagen),  PerfectPrep Endofree Plasmid Maxi Kit (5 PRIME, Inc.) and NucleoBond DNA Maxi Kit (MACHEREY-NAGEL GmbH & Co. KG).  The DNA quality was determined by measuring absorbance at 230, 260, 280 and 320 nm by spectrometry.The purity of DNA prepared by the three methods is as follows:MACHEREY-NAGEL > 5 Prime > Qiagen. Then pEGFP DNA was delivered with PolyJet™ (Cat # SL100688) DNA transfection reagent to NIH-3T3 cells per manufacturer’s protocol.  In accordance with DNA purity results, we found that DNA from MACHEREY-NAGEL gave the best transfection efficiency while Qiagen DNA gave the worst efficiency.  Therefore plasmid preparation kit from MACHEREY-NAGEL GmbH & Co. KG is confirmed to be the best choice to prepare tansfection grade DNA.