Introduction, Part 1.

1. General Characteristics


NUCLEOBOND AX is a silica-based, anion exchanger developed and manufactured by MACHEREY-NAGEL for routine separation of different classes of nucleic acids. It has an extraordinary high charge density on its hydrophilic, macroporous surface that results in a salt concentration range for binding and elution of nucleic acids that is much wider than on conventional anion exchangers. Thus it has a superior resolving power for separation of nucleic acids from one another and from proteins, carbohydrates, metabolites, and so on, with an efficiency not achieved by usual ion exchangers or by gel filtration.

The outstanding purity of this macroporous silica is guaranteed by production in our facilities and is an important prerequisite for the superior biological activity of the purified nucleic acids. It enables high mass recoveries by minimizing nonspecific adsorption effects. The impressive resolving power of NUCLEOBOND AX is illustrated in Figure 1.

Figure 1: Elution range of different substances on NUCLEOBOND AX Buffer: 100 mM Tris, 15% ethanol, adjusted to pH 6.3 with H3PO4 (phosphoric acid)

____________________________________________________________________________
Compound class      |         Salt concentration for elution
----------------------------------------------------------------------------
Plasmid DNA,
lambda DNA                                                           <----->

dsDNA (150 bp) <--->

ssDNA (M 13) <---->

mRNA <----------->

16S/23S rRNA <--->

5S RNA <----->

tRNA <----->

Bovine serum albumin <---->

Trinucleotides <---->

Proteins, dyes, <-----------> polysaccharides, etc. ---------------------------------------------------------------------------- KCl[M] | | | 0.5 1.0 1.5


 

2. NUCLEOBOND AX Cartridges

The NUCLEOBOND AX, ion exchange silica is secured between inert filter elements in polypropylene cartridges resulting in an inert and efficient separation tool. Five different sizes of NUCLEOBOND AX cartridges cover the whole purification range from nanogram amounts to 10 mg DNA or 40 mg RNA (see Table 1).

Table 1: Binding capacities of the cartridges

___________________________________________________________________________
NUCLEOBOND AX               Maximum Capacity/Run
cartridge type
---------------------------------------------------------------------------
AX 5                3 g lambda DNA, 5 g plasmid DNA, 20 g RNA
AX 20               12 g lambda DNA, 20 g plasmid DNA, 80 g RNA
AX 100              60 g lambda DNA, 100 g plasmid DNA, 400 g RNA
AX 500              300 g lambda DNA, 500 g plasmid DNA, 2,000 g RNA
AX 2000             1200 g lambda DNA, 2000 g plasmid DNA, 8,000 g RNA
AX 10,000           6000 g lambda DNA, 10000 g plasmid DNA, 40,000 g RNA
___________________________________________________________________________
No expensive instrumentation such as ultracentrifuges or HPLC equipment is needed to get a result at least as good as by CsCl gradients. All of the purified nucleic acids show absorption ratios of A260/A280 as high as 1.8-2.0. Low amounts of impurities, which often act as strong inhibitors for enzymatic processing, sequencing, transfections, transcriptions, and so on, are removed completely.

NUCLEOBOND AX cartridges are universal tools especially for the purification of DNAs. They can be used for the fast isolation of:

In addition, different types of RNAs can be purified including: Rapid procedures can be used for all these purifications on NUCLEOBOND AX cartridges. The usual time scale is minutes, not hours. Using the appropriate working procedures, oligonucleotides, RNA or different types of DNA can be purified on NUCLEOBOND AX with very high recoveries of over 90%. (Since the overall yield for the purification of cellular nucleic acids depends on variables like cell type, culture conditions, efficiency of cell lysis and so on, these factors may affect the results and should therefore be taken in consideration.)

All NUCLEOBOND AX types are resistant to organic solvents like alcohol, chloroform, and phenol. They are free of RNAse and DNAse.

Often there is not enough time to make up the whole set of buffers and other solutions needed for purifying a certain nucleic acid. In these cases NUCLEOBOND AX Kits should be used. They are the fastest, most reliable, and easiest way to get high quality nucleic acid in a high overall yield. NUCLEOBOND AX Kits are ready-to-use separation systems containing: cartridges, appropriate buffers (solutions and reagents), supports for individual cartridge, and complete protocols.

All separations are made in aqueous buffers with ethanol as a modifier. Low salt concentrations are required for the binding of the nucleic acid sample to this macroporous anion exchanger. The salt concentration is increased stepwise in the subsequent washing and elution steps. (Typical salt concentration for elution are listed in table 3). Tris-phosphate (buffer N) is used at pH 6.3. Other buffer systems may be used for special purposes. Adsorption of certain nucleic acids is possible from reaction mixtures or cell lysates between pH 5.0 to 7.0.

Table 3: Elution conditions for nucleic acids in different buffers

Buffer N: 100 mM Tris-phosphate, pH 6.3, 15% ethanol

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Eluting Compound             Concentration of KCl (mM)
------------------------------------------------------
                                  Buffer N
Proteins                          0-350
Nucleotides                       0-250
10-30 mer Oligonucleotides        250-500
tRNA                              450-650
rRNA                              950-1100
ssDNA                             1000-1150
dsDNA (>100 bp)                   1350-1500
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The salt concentration of elution of a given nucleic acid depends on the pH value of the eluant. That is why the pH of the buffers should be carefully controlled. A deviation of more than 0.1 pH unit from the given values will result in a change of salt concentration required for the elution of each nucleic acid. The basic rule of thumb is that at a higher pH, less salt is required to elute a particular nucleic acid type. (Figure 2)

Figure 2: Elution of nucleic acids depending on the pH of the buffer; 100 mM Tris-phosphate, 15% ethanol.

                 |      +
             1.5 |              +                          | 1.5
                 |       -              +                  | 
            1.25 |             -               +           | 1.25
                 |      #      #       -                   |  
KCl [M]      1.0 |                     #       -           | 1.0
                 |                             #           |     
            0.75 |      *      *       *                 + | 0.75
                 |                             *         - |
             0.5 |                                       # | 0.5
                 |                                       * |
                 +-----------------------------------------+
                        |       |       |       |       |
                pH     6.0     6.5     7.0     7.5     8.0

                  +=dsDNA    -=ssDNA    #=rRNA    *=tRNA
NUCLEOBOND AX can be used in the pH range 2.5 - 8.5 for about 3 hours without any change in its chromatographic properties. After that time the nucleic acids will elute at increasingly lower salt concentrations. The cartridges may be used at salt concentrations up to 2 M for elution. They are not damaged in the presence of denaturing agents like formamide or urea, nonionic detergents such as Triton X 100, and most organic solvents at temperatures up to 60C.

3. NUCLEOBOND AX Overview

NUCLEOBOND AX cartridges are easy to handle. Solutions are pipetted or just poured into the cartridges. The flow through the exchanger bed is by gravity. The cartridges do not run dry.

The easiest and most effective technique of passing buffer solutions through these cartridges is by gravity flow. All NUCLEOBOND AX cartridges may be used in this mode.

The polyethylene tubing supplied with each AX 2000 must be connected to the bottom of the cartridge before use to reach the optimum flow rate. All NUCLEOBOND AX cartridges may be stationed in test tubes or fixed by the use of clamps.

A simple and time-saving, four step procedure is used:

  1. Equilibration of NUCLEOBOND AX with the binding buffer
  2. Adsorption of the nucleic acids from the solution (cleared lysate, enzymatic reaction mixture, or extract from gels)
  3. Washing step (removes all contaminants)
  4. Elution of the desired nucleic acid
Equilibration step

Fill the appropriate volume of equilibration buffer into the cartridge. The buffer solution passes through the cartridge by gravity flow. If necessary, initiate the flow by means of a pipette or disposable syringe. The flow will stop when the surface of the buffer solution reaches the upper filter element. The remaining buffer assures an immediate start of the flow in all subsequent steps. It should not be pushed out.

Adsorption of the sample

The cleared sample should be loaded to the cartridge by gravity flow. The maximum flow rates and the flow by gravity are given for the different cartridges in table 4.

Washing step

It removes all contaminating substances. Washing is performed, in principle, like the equilibration step. Minimum volumes and maximum flow rates are given in table 4.

Elution of nucleic acids

Elution is carried out as above with 1 volume of elution buffer (see table 4) into a new tube. An additional amount of elution buffer equal to the dead volume of a cartridge can be added to maximize yield. The nucleic acid is routinely precipitated by the addition of 0.6-0.8 volumes of iso-propanol that has been equilibrated to room temperature. If only small amounts of nucleic acids are present, the pH should be adjusted to 5.2 before starting the precipitation step.

Table 4: Minimum volumes and maximum flow rates for the individual steps of the procedure

____________________________________________________________________________________
                           Minimum volume of buffer needed (ml)
Type of cartridge               AX        AX        AX        AX       AX
                                5         20        100       500      2000 
------------------------------------------------------------------------------------
Step of the procedure
1. Equilibration of cartridge   0.4       1.0       2.0       5.0      22
2. Binding of sample                 no restrictions 
3. Washing                      1.0       3.0       8.0       24       70
4. Elution of nucleic acid      0.4       0.8       2.0       6.0      22

Dead volume of cartridge 0.12 0.12 0.35 1.5 5.0 ------------------------------------------------------------------------------------ Maximum flow rate(ml/min.) Type of cartridge AX AX AX AX AX 5 20 100 500 2000 ------------------------------------------------------------------------------------ Step of the procedure 1. Equilibration of cartridge 0.5 1.0 2.0 6.0 18 2. Binding of sample 2.0 0.5 1.0 3.0 9 3. Washing 0.5 1.0 2.0 6.0 18 4. Elution of nucleic acid 0.2 0.5 1.0 2.5 6

Flow rate by gravity 0.2-0.5 0.4-0.8 0.6-1.3 1.5-3.0 4.5-7.5 ____________________________________________________________________________________

Attention!

The cartridges may be reused within 3 hours again without any change in their separation properties. Use a single cartridge for only one type of application, to prevent cross contamination.

Appropriate disposal precautions should be followed if the sample contains biohazards or radioactive material. NUCLEOBOND AX is not intended for use in humans or for clinical diagnosis.

Next , Introduction Part II: Performance Hints and Applications.

or Nucleobond AX properties and applications Index.


Copyright© 1998 The Nest Group, Inc. All rights reserved

Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

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Updated 1/20/98