How to prepare transfection grade plasmid DNA

Transfection efficiencies are affected by a variety of different parameters.  Besides factors such as cell culture, medium and vectors, one of the most critical parameters is DNA quality.  We prepared pEGF-N3 plasmid DNA with 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:
> 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 produced from the MACHEREY-NAGEL kit gave the highest transfection efficiency, while Qiagen's DNA preparation kit gave the lowest efficiency.  Therefore, the plasmid preparation kit from MACHEREY-NAGEL GmbH & Co. KG is confirmed to be the best choice to prepare transfection grade DNA.


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