Higher plasmid yields in less time compared to competitor anion exchange-based kits
When the yields and speed of NucleoBond Xtra Midi or Midi Plus kits and NucleoBond Xtra Maxi or Maxi Plus kits were compared to competitor anion exchange-based kits (Figure 2), the NucleoBond Xtra kits reduced preparation time by up to 60% and provided up to 100% higher yields than the competitor products.
Figure 2. Comparison of NucleoBond Xtra kits' yields and speed to those of competitor products. Plasmid DNA was isolated following each manufacturer's protocol using the maximum culture volume with high plasmid content. The yield of plasmid DNA was determined after DNA precipitation. NucleoBond Xtra and competitor kits including a desalting tool were compared in Panel A, left side (midi) and Panel A, right side (maxi), while kits without a desalting tool (requiring centrifugation for DNA precipitation) were compared in Panel B, left side (midi) and Panel B, right side (maxi). Note that the yields of plasmid DNA are slightly lower for kits including the desalting tool than for kits without the desalting tool, due to the residual DNA remaining on the tool.
Transfection
Higher transfection efficiencies than a competitor kit
NucleoBond Xtra Maxi and Product Q were each used to purify a 6-kb high-copy plasmid expressing gephyrin (a polypeptide associated with the postsynaptic receptor complex that plays a key role in the organization of the postsynaptic membrane) fused to an EGFP (enhanced green fluorescent protein) reporter. When each plasmid prep was transfected into HEK293 cells, which showed transient expression of gephyrin, higher transfection efficiencies were observed with NucleoBond Xtra Maxi than Product Q (Figure 3, compare Panels A and B).
Figure 3. Comparison of transfection efficiencies using plasmid DNA purified with NucleoBond Xtra Maxi and a competitor product. A 6-kb high-copy plasmid expressing gephyrin fused to an EGFP reporter was transfected into HEK293 cells after purification with NucleoBond Xtra Maxi (Panel A) or Product Q (Panel B). Plasmid purification from E. coli DH5α was performed according to each manufacturers' instructions using the maximum culture volume with high plasmid content. NucleoBond Xtra Maxi yielded 1,222 μg of plasmid with an A260/280 of 1.84. Product Q yielded 557 μg of plasmid with an A260/280 of 1.86. HEK293 cells in 12-well plates (2.5 x 104 cells/well) were transfected for 48 hr with 0.1–0.25 μg/well of plasmid DNA using FuGENE HD/6 (Roche, 1.5 μl/well). Data kindly provided by Prof. Dr. Guenter Schwarz, Institute of Biochemistry, University of Cologne, Germany.
Sensitive cells
Transfection of highly sensitive primary neurons
NucleoBond Xtra EF was used to purify a 9-kb high-copy plasmid expressing neuroligin (a postsynaptic transmembrane protein) fused to a GFP reporter. When the purified plasmid was transfected into primary rat hippocampal neurons, which are highly sensitive to endotoxins, fluorescence microscopy showed transient expression of neuroligin (Figure 4).
Figure 4. Transfection of primary rat hippocampal neurons using plasmid DNA purified with NucleoBond Xtra EF. A 9-kb high-copy plasmid expressing neuroligin fused to a GFP reporter was transfected into primary rat hippocampal neurons after purification with NucleoBond Xtra EF. Plasmid purification from E. coli XL1-Blue was performed according to the manufacturer's instructions. Primary rat hippocampal neurons were cultured in vitro for six days and transfected with 1 µg of plasmid DNA per well using calcium phosphate. The neurons were visualized by fluorescence microscopy 48 hr post-transfection. Data kindly provided by Dr. Nina Wittenmayer, Ruprecht-Karls-University Heidelberg, Institute for Anatomy and Cell Biology, Dept. of Medical Cell Biology, Heidelberg, Germany.
Endotoxin-free DNA for transfection of primary rat hippocampal neurons
A 4.5-kb high-copy plasmid purified using NucleoBond Xtra Maxi Plus EF was shown to transfect primary rat hippocampal neurons using fluorescence microscopy, as seen in both panels of Figure 5.
Figure 5. Endotoxin-free DNA for transfection of primary rat hippocampal neurons. A 4.5-kb high-copy plasmid was transfected into primary rat hippocampal neurons after purification from E. coli DH5α with NucleoBond Xtra Maxi Plus EF. Primary rat hippocampal neurons were cultured in vitro for 14 days and transfected with 1 µg of plasmid DNA per well using NeuroMag transfection reagent (OZ Biosciences). The neurons were visualized by fluorescence microscopy 48 hr post-transfection. Data kindly provided by OZ Biosciences, Marseille, France.
Protocol
No extra incubation is needed for endotoxin removal
The NucleoBond Xtra EF procedure, which is very similar to the NucleoBond Xtra procedure, requires no extra incubation step for endotoxin removal (Figure 6).
Figure 6. Comparison of the NucleoBond Xtra and NucleoBond Xtra EF procedures.
