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Tech Note

NucleoBond Xtra Midi/Maxi/EF

A new generation of anion exchange plasmid purification kits—ultra-fast, endotoxin-free plasmid DNA with high yields

  • Plasmid DNA containing less than 0.05 EU/μg—transfect highly sensitive cells
  • Patented endotoxin removal via an additional washing step—no extra ice incubation needed
  • Optimized column design—midipreps in approximately 45 min, maxipreps in 50 min
  • Column filter included—high filter flow rates, lysate clearing and column loading in parallel
  • Improved silica matrix—typical yields of 400 μg (midi) and 1,000 μg (maxi)
  • NucleoBond Xtra Plus EF kits contain NucleoBond Finalizers to speed up DNA precipitation
NucleoBond Xtra Combi Rack for maxi and midi columns

Figure 1. The NucleoBond Xtra Combi Rack holds both maxi and midi columns.

Yield and prep time  

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.

NucleoBond Xtra kit yields and speed vs. a competitor

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).

Transfection efficiencies with NucleoBond Xtra Maxi vs. a competitor

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).

NucleoBond Xtra Maxi EF-purified plasmid transfects primary neurons

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.

Endotoxin-free DNA transfects primary rat hippocampal neurons

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).

Comparing NucleoBond Xtra and NucleoBond Xtra EF procedures

Figure 6. Comparison of the NucleoBond Xtra and NucleoBond Xtra EF procedures.

Related Products

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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NucleoBond Xtra Midi EF (50) 50 preps for the isolation of endotoxin-free plasmid DNA - NucleoBond Xtra Midi Columns, NucleoBond Xtra Midi Column Filters, buffers, RNase A

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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740422.10 NucleoBond® Xtra Midi Plus EF 10 Preps $194.00 *

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Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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740422.50 NucleoBond® Xtra Midi Plus EF 50 Preps $840.00 *

NucleoBond Xtra Midi Plus EF (50) 50 preps for the isolation of endotoxin-free plasmid DNA - NucleoBond Xtra Midi Columns, NucleoBond Xtra Midi Column Filters, NucleoBond Finalizers, syringes, buffers, RNase A

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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