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.

 


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