Introduction, Part II

Summary of buffer sequence for different Nucleobond AX and AXG applications

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Applications                 Equilibration of  Adsorption of   Washing   Elution of
                             the cartridge     sample [KCl]    step      nucleic acid
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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
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If CsCl purity is not required, or you're not doing automated sequencing, consider using NucleoTrap and NucleoTraPCR for final volumes <50µl or NucleoSpin and NucleoSpin Extract for final volumes >50µl 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