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。转染效率的高低与DNA的质量相符,由MACHEREY-NAGEL纯化DNA对应的转染效率最高,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 DNA
Transfection 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.