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).
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.
|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)
|< 300 kbp||< 300 kbp|
|Typical yield||400 µg||1000 µg|
Xtra Midi Plus
Xtra Maxi Plus
|Preparation time||70 min/prep||30 min/prep||75 min/prep||35 min/prep|