Purification of Genomic DNA from Cell Cultures and Tissues:

Proteinase K Procedure Using a Plasmid or a Lambda Kit (1)

This procedure using Nucleobond AX cartridges allows one to purify genomic DNA with fragments up to 70 Kb in size. For larger fragments, in the 200-300 Kb range, the AXG tubes should be used. (Please read the entire protocol before starting the prep.)

Special conditions for the type of NUCLEOBOND AX cartridge used
Step of the procedure AX 20 AX 100 AX 500 Amount of cells/mass of tissue used 4 x 10^4 2 x 10^5 1 x 10^6 /20 mg /100 mg /0.5 g 1. Pulverize the material in liquid 0.5 ml 2 ml 8 ml nitrogen prior to adding it to the K1+ lysis buffer or homogenize the material directly in the K1+ lysis buffer (not included in the plasmid kit). Incubate at 50-60C for 1-2 hours preferably with gentle shaking.

2. Add 1/2 volume of cold buffer S3 250 Ál 1 ml 4 ml and mix gently. Pellet the SDS and debris by centrifugation at 4C and 12,000 x g for 10-20 minutes.

3. Transfer the supernatant to a 11 mg 45 mg 180 mg fresh tube, add 15 mg/ml KCl to a final concentration of 200 mM. Adjust the pH to 6.3 as required with dilute phosphoric acid. (Optional)

4. Equilibrate a cartridge with 1 ml 2 ml 5 ml buffer N2 and load the sample. (Substitute L2 if using a lambda kit.)

5. Wash the cartridge with buffer N3. 3 x 1 ml 3 x 4 ml 3 x 12 ml (Substitute L3 if using a lambda kit.)

6. Elute the DNA with buffer N5. 1 ml 3 ml 6 ml (Substitute L5 if using a lambda kit.) (High GC content)

7. Add 0.8 volumes of room temp. 0.8 ml 2.4 ml 4.8 ml iso-propanol to the eluate.Incubate on ice for 15 minutes and centrifuge at 15,000 x g for 10-20 min. at 0C.

8. Wash the DNA pellet with 70% yes yes yes ethanol, air dry briefly,and dissolve in an appropriate buffer for further handling.

1. This procedure will only work well on the cartridges if one's genomic DNA fragments are not much larger than 70 Kb. (For most procedures requiring genomic DNA including PCR, restriction enzyme digestion and Southern blot analysis, lambda or cosmid library construction, 70 Kb fragments are more than sufficient.) If genomic DNA fragments larger than 70 Kb are required, then Nucleobond AXG tubes, which contain loose resin in contrast to resin secured between membranes, should be used.

2. Optional: When purifying genomic DNA from plant samples, an extra step can be added at this point to reduce the level of polysaccharides in a prep prior to loading it onto a cartridge. This procedure is adapted from the one described by Micheals et al. (Michaels, S. D., M. C. John, and R. M. Amasino. 1994. Removal of Polysaccharides from Plant DNA by Ethanol Precipitation. BioTechniques 17:274-276.) and is performed as follows:

(a) Add 0.35 volumes of 100% ethanol to the solution in step 3 above prior to adjusting the pH.

(b) Mix and incubate on ice for 15-20 minutes. Centrifuge at 10,000 x g and 4C for 5-10 minutes.

(c) Titrate the pH of the supernatant to 6.3 and load it onto an equilibrated Nucleobond AX cartridge and continue from step 5 above.

3. 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.

Buffer solutions Storage K1+ - 50 mM Tris-HCl, pH 8.0, 50 mM KCl , 10-20 mM EDTA, -20C 1 mM dithiothreitol,0.5% SDS, 100 mg/ml RNAse A, 200 mg/ml proteinase K (RNAse A should be added only just prior to use from a 10 mg/ml stock) S3 - 2.8 M K-Acetate, pH 5.2 4C N2 or L2 - 100 mM Tris, 15% ethanol and 900 mM KCl RT adjusted with phosphoric acid to pH 6.3 N3 - 100 mM Tris, 15% ethanol and 1150 mM KCl adjusted RT with phosphoric acid to pH 6.3 L3 - 100 mM Tris, 15% ethanol and 1000 mM KCl adjusted RT with phosphoric acid to pH 6.3 N5 or L5 - 100 mM Tris, 15% ethanol and 1000 mM KCl RT adjusted with phosphoric acid to pH 8.5

To make your own buffers or for more information please refer to N and L Series Buffer Preparation, N5 and L5 Buffer Preparation, and Nucleobond AX Buffers.

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

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Updated 1/20/98