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NucleoBond® Xtra Midi /Maxi

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MACHEREY-NAGEL has developed NucleoBond® Xtra, a new generation of anion exchangers based on the patented NucleoBond® technology.

With NucleoBond® Xtra typical yields of ≥ 250 μg (Midi) or ≥ 1000 μg (Maxi) of ultra-pure plasmid DNA can be obtained in about half of the time compared to other Midi and Maxi kits based on anion-exchange chromatography.


 

NucleoBond® Xtra Midi and Maxi kits contain enlarged columns which leads to lower silica resin beds. This in turn enables faster flow of lysate and buffers through the columns. Specially designed column filters are included for convenient and time-saving clarification of bacterial lysates. The column filters are supplied inserted in the NucleoBond® Xtra columns and allow parallel clarification of bacterial lysate and loading onto the column. Their large, structured surface leads to high filter flow rates and minimized risk of clogging. The improved silica material with greater DNA binding capacity is based on the proven NucleoBond® anion-exchanger group MAE, methylaminoethanol (patented technology). Optimized buffer compositions additionally lead to improved alkaline lysis and increased column flow rates.

 

Bacteria are harvested from an overnight culture and lysed by an optimized alkaline lysis procedure. The bacterial lysate is cleared and loaded onto the equilibrated column in one step (plasmid DNA binds to the anion-exchange resin). The first washing step using equilibration buffer is performed with inserted column filter to wash out residual lysate from the filter and obtain maximum recovery of DNA. After subsequent washing, the purified plasmid DNA is eluted in a high-salt buffer and precipitated with isopropanol. Precipitation can be performed by centrifugation (NucleoBond® Xtra Midi or Maxi) or by using the NucleoBond® Finalizer / Finalizer Large included in the NucleoBond®Xtra Midi Plus/Maxi Plus kits to reduce the total prep time to about 30 minutes (Midi Plus) or 35 minutes (Maxi Plus).

NucleoBond Xtra Procedure


LyseControl — for highest yields

The new LyseControl, included in the NucleoBond® Xtra procedure, visualizes a complete neutralization for efficient alkaline lysis. LyseControl is a blue reagent premixed for your convenience with Lysis Buffer LYS. Upon addition of lysis buffer to the resuspended cells (Fig. A), the cell suspension turns blue (Fig. B). The blue solution is gently inverted 5 times and incubated for 5 min to avoid contamination with genomic DNA. Upon addition of Buffer NEU (Fig. C) to neutralize the lysate, LyseControl turns colorless. Traces of blue LyseControl (Fig. D) indicate an insufficient mixing. This step removes SDS, renatures plasmid DNA, and precipitates proteins and genomic DNA. Hence, it prevents filterclogging, maximizes plasmid DNA yield, and minimizes contamination.

 

 A    Cell pellet in resuspension buffer (RES)
 B    Addition of lysis buffer (LYS – including LyseControl) to cell suspension. The cell suspension turns blue.
 C    Addition of neutralization buffer (NEU).
LyseControl starts turning colorless
start mixing.
 D    Traces of blue LyseControl still visible
insufficient mixing.
 E    LyseControl turned completely colorless
sufficient mixing.
Technical Data:

NucleoBond® Xtra Midi NucleoBond® Xtra Maxi
Technology Anion-exchange chromatography Anion-exchange chromatography
Format Midi gravity flow columns Maxi gravity flow columns
Lysate clarification Column filters Column filters
Sample material < 200 mL (high copy)
< 400 mL (low copy)
< 600 mL (high copy)
< 1200 mL (low copy)

Vector size

< 300 kbp < 300 kbp
Typical yield 400 µg 1000 µg
A260/A280 1.80–1.95 1.80–1.95
Isopropanol precipitation

Xtra Midi

Centrifugation

Xtra Midi Plus

NucleoBond®Finalizer

Xtra Maxi

Centrifugation

Xtra Maxi Plus

NucleoBond®Finalizer Large

Preparation time 70 min/prep 30 min/prep 75 min/prep 35 min/prep


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