Purification of Genomic DNA From Prokaryotes

Working procedure                          AXG 20           AXG 100
Culture volume:                               5-10 ml           25-50 ml
Maximum amount of genomic DNA:                10-20 g          150-200 g

1.The RNAse must be added to buffer S1 0.3 ml 1.5 ml before use. S1 should be stored at 4C. Resuspend the bacterial pellet/cells in buffer S1.

2. Add the freshly prepared lysozyme 50 l 200 l solution (10 mg/ml in 10 mM Tris-HCl; pH 8.0) and incubate for 45 min. at 37C. Note: Lysozyme will not work efficiently at less than pH 8.0.

3. Add KCl from a 2.0 M stock solution 90 l 440 l to a final concentration of 0.4 M and 10 l 60 l add water to bring to final volume.

4. Add an equal volume of phenol 450 l 2.2 ml equilibrated with Tris buffer (pH 8.0). Mix by inverting the tube until a complete emulsion has formed. Separate the phases by centrifugation for 5 min. at 3000 x g. Transfer the aqueous phase into a clean tube.

5. Extract the aqueous phase with an equal ~ 400 l ~ 2.0 ml volume of 24:1 chloroform/iso-amyl alcohol. Transfer the aqueous phase into a clean tube.

6. Add proteinase K (20 mg/ml in H2O) and 5 l 20 l incubate the mixture for 60 minutes at 55C. Note: This step is optional.

7. Check that the pH is not greater yes yes than 6.3. If it is, titer it as required with dilute H3PO4.

Equilibration of the NUCLEOBOND AXG tube 8. Add buffer N2 to an AXG tube and 1 ml 5 ml invert the tube for 2 min. on an end-over- end mixer at 10 rotations per minute (or manually).

9. After a short centrifugation step (100-200 rpm, 2 min.) the supernatant is discarded.

10. Add buffer N3 to the sample containing 600 l 1 ml the genomic DNA. Incubate the AXG tube for 10-15 min. on an end-over-end mixer at 10 rotations per minute or manually. After a short centrifugation step (100-200 rpm, 2 min.) the supernatant is discarded.

11. Wash the AXG tube with buffer N3, 2 x 1 ml 2 x 5 ml centrifuge, and discard the supernatant.

12. Elute the genomic DNA with buffer N5. 1 ml 3 ml Incubate the tube for 5 min. on an end- over-end mixer at 10 rotations per minute or manually. Centrifuge and transfer the supernatant in a clean tube. (High GC content)

13. Precipitate the DNA by adding 0.8 0.8 ml 2.4 ml volumes of room temperature iso-propanol. Incubate for 15 min. on ice and centrifuge at maximum speed (4C; 5-10 min.).

14. Dissolve the DNA gently overnight at 4C in TE or water.

SEE ALSO: NucleoSpin BLOOD (for more info email us)

Solutions Storage: Lysozyme (10 mg/ml in mM Tris-HCl; pH 8.0) -20C (in aliquots) Phenol (50 mM Tris-HCl; pH 8.0) RT Chloroform: Iso-amyl alcohol (24:1) RT Proteinase K (20 mg/ml in H2O) 4C S1 - 50 mM Tris-HCl, 10 mM EDTA, 100 g RNAse A/ml; pH 8.0 4C N2 - 100 mM Tris-phosphate, 900 mM KCl, 15% Ethanol, pH 6.3 RT N3 - 100 mM Tris-phosphate, 1150 mM KCl, 15% Ethanol, pH 6.3 RT N5 - 100 mM Tris-phosphate, 1000 mM KCl, 15% Ethanol, pH 8.5 RT

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

Attention: In order to minimize mechanical shearing of genomic DNA, only use wide-bore or cut-off pipette tips.

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.

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

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Updated 1/20/98