Introduction, Part II

4. Nucleobond AX Applications and Performance Hints

Summary of buffer sequence for different Nucleobond AX and AXG applications

Applications                 Equilibration of  Adsorption of   Washing   Elution of
                             the cartridge     sample [KCl]    step      nucleic acid
  • Preparation of E. coli buffer N2 850 mM buffer N3 buffer N5 plasmid DNA including P1 constructs and BACs (bacterial artificial chromosomes)

  • Preparation of phage buffer L2 850 mM buffer L3 buffer L5 lambda- DNA

  • Preparation of single- buffer L1 400 mM buffer L2 buffer L5 stranded phage M13 DNA

  • Purification of total RNA buffer R1 350 mM buffer R1 buffer R3

  • Removal of linker DNA buffer N2 850 mM buffer N3 buffer N5 from DNA fragments (>100 base pairs) in cloning experiment

  • Removal of nucleotides buffer N1 500 mM buffer N2 buffer N5 from DNA probes (>60 bp) after labeling reactions

  • Purification of DNA buffer N2 850 mM buffer N3 buffer N5 restriction fragments (>100 base pairs) after elution from gels

  • Quick removal of enzymes buffer N1 400 mM buffer N2 buffer N5 (restriction endonucleases polymerases, phosphates, etc.) from DNA (>60 bp)

  • Purification of PCR buffer N2 850 mM buffer N3 buffer N5 products (>100 bp)

  • Removal of nucleotides buffer U1 200 mM a. buffer U2 buffer U3 from oligos (>20 mer) after b. buffer U0 labeling reactions

  • Purification of genomic DNA buffer N2 850 mM buffer N3 buffer N5 _____________________________________________________________________________________
  • If CsCl purity is not required, or you're not doing automated sequencing, consider using NucleoTrap and NucleoTraPCR for final volumes <50Ál or NucleoSpin and NucleoSpin Extract for final volumes >50Ál since no precipitation step will be necessary.

    The yield of plasmid DNA purified with Nucleobond AX is dependent on media, host vector as well as type and size of plasmids. XL1 blue cells with blue script plasmid have a high copy number. One must use a larger volume of lysing buffer or a lower volume of culture to obtain the best results. Other high copy plasmids are: pUC, pBluescript, pGEM, etc. ; while low copy plasmids and cosmids are: pBR 322, etc..

    If low copy number plasmids like pBR 322, cosmids, or P1 constructs, PACs or BACs are to be purified, larger culture volumes may have to be processed in order to maximize yields. To reduce the amount of cellular material contaminating the supernatant of step 3 of the Plasmids and Cosmids Protocol and to ensure good flow rates of the cartridge in step 5, the buffer volumes for S1, S2 and S3 must be increased in relation to higher culture volume or cell density. Refer to P1 Constructs and Other Low Copy Number Plasmids, Cosmids, PACs and BACs for required lysis buffer volumes and further details.

    The Nucleobond AX procedure is optimized for cultures grown in LB-media. If rich media like TB-media (terrific broth) are used, the volumes of the buffers S1,S2 and S3 have to be increased 2 fold. Furthermore, one additional washing step with buffer N3 is recommended.

    Table 6. Expected yields from indicated volume of media.

    High copy no.   Low copy no.     Type          Expected
    Volume LB       Volume LB        Cartridge     Yield
     1-5 ml             3-20 ml             AX 20           3-20 g
     5-50 ml            20-100 ml           AX 100          20-100 g
     50-200 ml          100-500 ml          AX 500          100-500 g
     200-500 ml         500-2000 ml         AX 2000         500-2000 g
     500-2000 ml        2000-10000 ml       AX 10000        2000-10000 g

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

    Back to the Nucleobond AX applications guide

    Updated 1/20/98