A Nuclease Free Procedure for the Purification of Bacteriophage lambda DNA and Other Single- and Double-Stranded Viral Nucleic Acids (footnote)

This protocol describes the purification of lambda DNA from plate or liquid lysates, although plate lysates tend to yield more consistent results. Volumes ranging from 1 ml to 250 ml can be processed using the corresponding NUCLEOBOND AX cartridge. Bacterial nucleic acids are separated with a specially activated matrix. An approximate correlation between cartridge size, culture volume and the amount of DNA is given in the table below.

Note: Before starting with the isolation of Lambda DNA prepare buffer T2.

T2: The PEG/NaCl mixture should be dissolved in distilled water by stirring. Heating the solution may facilitate this process. The T2 buffer should be stored at 4C and used cold.

T4:Dissolve precipitated SDS by warming to 40C before use, if necessary.

NOTE: All volumes given in the protocol are minimum volumes. The yield of lambda DNA depends on several properties like E. coli strain, particular lambda vector, titer of the lambda lysate, efficiency of inoculation, and culture conditions.

Procedure                                    Type of Cartridge
                                       AX 20       AX 100      AX 500
     Maximum culture volume                   10 ml         50 ml         250 ml
     Maximum amount of lambda DNA             12 g         60 g         300 g

Equilibration of the extraction matrix: 1. Add CHCl3 (1 l/ml) to the lysate, mix, and centrifuge (6000 rpm; 10 min; 4C). Transfer the supernatant to a clean tube.

2. Add 30 mg KCl/ml lysate in order to adjust a 0.4 M final concentration of KCl for the lysate.

3. Adjust the pH of the solution with "diluted" phosphoric acid (H3PO4)to pH 6.3. ATTENTION: phosphoric acid is a strong acid. For the preparation of diluted phosphoric acid add 3-4 drops to 2 ml of water.

4. Resuspend the extraction matrix 0.5g of 2.5g of 10g of in buffer L1 and mix thoroughly matrix w/ matrix w/ matrix w/ (~5 min.) Use for the given cartridges 5 ml L1 10 ml L1 30 ml L1 the following amounts:

5. Centrifuge (greater or equal to 6000 rpm; 5 min.; RT) and discard the supernatant.

6. Add the lysate to the extraction matrix and mix gently by inverting the tube (~10 min.) manually or with an end over end mixer.

7. Pellet the extraction matrix by centrifugation (5000 rpm; 5 min.; RT) and transfer the supernatant to a new tube. Attention: Repeat this step if residual extraction matrix is visible.

Preparation of the lambda particles: 8. Add buffer T2 (4C), mix thoroughly, 3 ml 15 ml 75 ml and incubate on ice for 60 min.

9. Centrifuge at greater than or equal to yes yes yes 10,000 x g at 4C for 10 min. Discard the supernatant and drain the pellet by standing the tube in a tilted position for 3 min.

10. Resuspend the pellet by pipetting 0.5 ml 1.5 ml 5 ml buffer T3 several times along the wall of the tube. (note #2)

11. Add buffer T4, mix gently, and 0.5 ml 1.5 ml 5 ml incubate at 65C for 10 minutes. Following this, put on ice for 5 minutes.

12. Add buffer S3 (pre-cooled 4C), mix 0.5 ml 1.5 ml 5 ml immediately by inverting the the tube 6-8 times, and centrifuge (greater or equal to 12,000 x g) at 4C for 35 min.

Isolation of lambda DNA: 13. Equilibrate a NUCLEOBOND AX cartridge 1 ml 3 ml 8 ml with buffer L2. (For single-stranded DNA viruses such as M13, substitute buffer L1 in this step)

14. Transfer the clear supernatant of yes yes yes step 12, immediately after centrifugation onto the cartridge.

15. Wash the contaminating substances 2 x 1 ml 2 x 2.5 ml 2 x 5 ml from the cartridge with buffer L3. (For single-stranded viruses, substitute buffer L2 in this step.)

16. Elute the DNA from the cartridge with 1 ml 2.5 ml 6 ml buffer L5. (note #3)

17. Add about 0.75 volumes iso-propanol 0.75 ml 1.9 ml 4.5 ml equilibrated to room temperature.

18. Collect precipitated lambda DNA by centrifugation at 12,000 x g for 30 minutes and wash the pellet with 70% ethanol.

Buffer solutions: Storage: T2: 25% polyethylene glycol (PEG 6000), 2.5 M NaCl 4C T3: 50 mM Tris-HCl, 150 mM NaCl, 30 mM EDTA, pH 7.7 RT T4: 2% SDS RT S3: 2.8 M K-acetate, pH 5.2 RT L1: 100 mM Tris-phosphate, 400 mM KCl, pH 6.3, 15% Ethanol RT L2: 100 mM Tris-phosphate, 900 mM KCl, pH 6.3, 15% Ethanol RT L3: 100 mM Tris-phosphate, 1000 mM KCl, pH 6.3, 15% Ethanol RT L5: 100 mM Tris-phosphate, 1000 mM KCl, pH 8.5, 15% Ethanol RT

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

NOTE 1: This yield of DNA from this procedure is very titer dependent. In addition, the minimum titer for one virus may not specifically apply to another. For example, the minimum recommended titer for phage lambda is about 4-5 x 10^9 pfu/ml. At this level one can expect to achieve a maximum theoretical yield of 200-250 ng lambda DNA per ml of lysate. However, for a 3 Kb single-stranded phagemid, the minimum recommended titer would be about 1-2 x 10^11 pfu/ml. At this level one can expect to achieve a maximum theoretical yield of 150-300 ng phagemid DNA per ml of lysate. The calculations below for molecular weight use 330 daltons per nucleotide of single-stranded DNA and 660 daltons per base pair of double stranded DNA.

  1. Double-Stranded Lambda DNA Size 50 Kb; MW = 50000 x 660 = 3.3 x 10^7 g/mole or 3.3 x 10^13 g/mole

    Thus, at a titer of 5 x 10^9 the maximum amount of DNA per ml that can be purified can be calculated as:

    (5 x 10^9 pfu/ml / 6.02 x 10^23 /mole) x (3.3 x 10^13 g/mole) = 1.65 / 6.02 ..approx.. 0.27 g/ml

  2. Single-Stranded Phagemid Size 3 Kb; MW = 3000 x 330 ..approx.. 1 x 10^6 g/mole or 1 x 10^12 g/mole

    Thus, at a titer of 1 x 10^11 the maximum amount of DNA per ml can be calculated as: (1 x 10^11 pfu/ml / 6.02 x 10^23 /mole) x (1 x 10^12 g/mole) = 1 / 6.02 ..approx.. 0.17 g/ml

NOTE 2: If the lambda DNA consistently exhibits some degree of partial degradation as a result of exposure to endonuclease A from the plating bacteria (as visualized by some smearing below the lambda DNA band on an agarose gel), then add proteinase K to a final concentration of 200 g/ml to the incubation in step 11 above.

NOTE 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.

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

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Updated 1/20/98