Purification of Plasmids and Cosmids

A Modified Alkaline Lysis Procedure

Using this modified method of Birnboim and Doly, bacterial cells are lysed by a NaOH/SDS solution. Chromosomal and plasmid DNA are partially denatured under these alkaline conditions. It is important to control the duration of this denaturing step thoroughly. The following addition of potassium acetate is a crucial step as well. It results in a precipitate containing the chromosomal DNA and other cellular compounds. Plasmid DNA stays in solution and is purified to homogeneity on the corresponding NUCLEOBOND AX cartridge. For buffer volumes and operation conditions see the table below. Please read the remarks before you start your prep.

Special conditions for the type of NUCLEOBOND AX cartridge used

Step of the procedure AX-5 AX-20 AX-100 AX-500 AX-2000 AX-10^4 Culture volume Volumes. 1-5ml 1-10ml 10-100ml 100-500ml 500-2000ml 2-10L (A600 approx. 1 O.D.)

Maximum amount of plasmid DNA 5 g 20 g 100 g 500 g 2000 g 10000 g

Cell disruption: 1. Carefully resuspend 0.4 ml 0.4 ml 4.0 ml 12 ml 35 ml 80 ml the bacterial cell pellet in buffer S1.

2. Add the appropriate 0.4 ml 0.4 ml 4.0 ml 12 ml 35 ml 80 ml volume of buffer S2, mix gently by inverting the tube, and incubate at room temperature or 5 min. Do not vortex to prevent the release of chromosomal DNA from the cell debris.

3. Add buffer S3 and mix 0.4 ml 0.4 ml 4.0 ml 12 ml 35 ml 80 ml gently by inverting 6-8 times until a homogeneous suspension is formed. Incubate on ice for 5 minutes and centrifuge at high speed (>12,000 x g)at 4C for: 20 min 20 min 38 min 45 min 60 min 60 min

Equilibration: 4. Equilibrate the cartridge 1.0 ml 1.0 ml 2.0 ml 5 ml 22 ml 100 ml with buffer N2

Adsorption: 5. Carefully remove the yes yes yes yes yes yes supernatant from the white precipitate and load onto an equilibrated NUCLEOBOND AX cartridge

Wash: 6. Wash the cartridge with 3x1 ml 3x1 ml 2x4 ml 2x12 ml 2x35 ml 2x100 ml buffer N3.

Optional: 7. Wash the 3x1 ml 3x1 ml 3x4 ml 3x12 ml 3x35 ml 3x100 ml cartridge with the E2B endotoxin removal buffer.

Elution: 8. Elute plasmid DNA with 0.8 ml 0.8 ml 2.0 ml 6 ml 22 ml 100 ml buffer N5

If this elution step is repeated one additional time, up to 30% more DNA can be isolated. This is especially true for the AX-100 and AX-500 cartridges. Note the flow rates of Nucleobond AX cartridges are up to two times faster than for Qiagen Tip cartridges which means that clogging from higher viscosity solutions is less likely (less sensitive to cell density problems), and the amount of losses from endonucleases will be lower due to shorter contact times.

(For high GC content DNA, see optional elution for high GC content)

9. Precipitate the DNA by      0.6 ml  0.6 ml   1.4 ml    4.2 ml   16 ml      70 ml
adding 0.7 ml volumes of room 
temperature iso-propanol.
Centrifuge at >12000 x g and 
4C for 10-20 minutes.  Wash 
the pellet with 70% ethanol, 
air dry briefly (about 5 min.), 
and dissolve in an appropriate 
buffer for further manipulations.

Buffer Solutions Storage S1 - 50 mM Tris-HCl, 10 mM EDTA, 100 g RNAse A / ml, pH 8.0 4C S2 - 200 mM NaOH, 1% SDS RT S3 - 2.8 M K-Acetate, pH 5.2 4C N2 - 100 mM Tris, 15% ethanol and 900 mM KCl adjusted with phosphoric acid to pH 6.3 RT N3 - 100 mM Tris, 15% ethanol and 1150 mM KCl adjusted with phosphoric acid to pH 6.3 RT N5 - 100 mM Tris, 15% ethanol and 1000 mM KCl adjusted with phosphoric acid to pH 8.5 RT

*This salt concentration is sufficient for the elution step, because of the increased pH value of this buffer.

To make your own buffers or for more information please refer to Buffer Preparation and Nucleobond AX Buffers.

For further suggestions please see Special Conditions for Purifying P1 Constructs and Other Low Copy Number Plasmids, Cosmids, PACs and BACs or see Special Conditions for Plasmid Preparation from Gram-Positive Bacteria.

Remarks: The extra tubing supplied with each AX-2000 cartridge must be attached to achieve the optimal flow rate.

For high GC content DNA: elute with a buffer of 50% formamide, 1.0 M KCl, 15% EtOH & 0.1 Tris-phosphate, pH 8.5, heated to 60C before loading. For the recommended procedure for preparing this buffer refer to N5 and L5 Elution Buffer Preparation. Precipitate the DNA with an equal volume of 70% (not 0.7 vol) isopropanol at room temperature and centrifuge at high speed (>12,000 x g) at 4C. The DNA has to be washed with 70% isopropanol to remove most of the formamide. A second washing step with 70% ethanol can be performed. After a short drying step DNA can be dissolved in an appropriate buffer for further manipulations.

If the plasmid DNA needs to be purified rapidly on a small scale and absolute purity is not required, then an alternative product, NucleoSpin, should be used. The latter kit consists of a silica membrane in a spin column format and DNA binding is performed using chaotropic salts. Elution is accomplished with a low ionic strength buffer such as TE. Please refer to the NucleoSpin and NucleoTrap Properties and Applications Guide for further details on this kit.

The RNAse (already heat treated and DNAse-free), must be added to buffer S1 before use. S1 should be stored then at 4C. The SDS in buffer S2 will precipitate at temperatures below 20C. If this is the case, store the bottle for a few minutes at about 30C to 40C and mix well and equilibrate to room temperature before use. The SDS is removed by buffer S3 (white precipitate) and will not be loaded on the cartridge. This step is very important! Take care that the supernatant of step 5 is clear! SDS will clog the cartridge and prevent the adsorption of nucleic acids.

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..

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
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 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 Special Conditions for Purifying P1 Constructs and Other Low Copy Number Plasmids, Cosmids, PACs and BACs for required lysis buffer volumes and other details.

The following suggestions are offered for situations when rather small amounts of DNA are to be purified (<0.5 g).

(A) Alternative column washing, elution, and precipitation:

  1. Use the smallest type of cartridge, which can bind your sample. Equilibration of the cartridge, adsorption of sample and washing should be done as usual.
  2. Wash the cartridge with 0.5 M Na-acetate, 15% ethanol (pH 7.0). Use about 50% of the volume given for step 6 above.
  3. The DNA is eluted by 0.05 mM Tris-HCl containing 1.0 M sodium acetate (pH 8.5). Use the volumes given for step 7. The eluate should be brought to acidic pH by 0.5 M sodium acetate (pH 4.5). Glycogen (20 g/ml) should be added as carrier (Heims, C. et al. 1985. DNA 4:39).
  4. Precipitation of the nucleic acid is done with 2 or 2.5 volumes of ethanol at -20C for 1 hour.
  5. The centrifugation step (>15000 x g, 1 hour) should be done at 0C or at a lower temperature.
(B) Use ultrafiltration instead of precipitation to remove salt.

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

Back to the Nucleobond AX applications guide

Updated 1/20/98