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Large Scale Extraction of Filamentous Bacteriophage Replicative Form (RF) DNA
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol describes the purification of the double-stranded replicative form (RF) bacteriophage DNA from two 1-liter cultures of stationary phase bacterial cells harboring an fd-tet based plasmid (see Hint #2). RF DNA is synthesized after transfer of a single stranded DNA (ssDNA) genome from a bacteriophage particle to a bacterial cell. The ssDNA is converted into RF DNA by synthesis of a complementary strand to the ssDNA by host cell DNA polymerase. The RF DNA then replicates further as a plasmid, and serves as the template for synthesis of new ssDNA molecules that are packaged and released from the infected cell as phage particles.
Procedure
VERY IMPORTANT NOTE: If you are preparing RF DNA from the frameshifted vectors fUSE1, fUSE3 or fUSE5, see Hint #3 before starting this protocol.
1. Inoculate two 1-liter cultures of NZY Medium with 1 ml per culture of a saturated overnight culture of bacterial cells carrying an fd-tet-based vector (see Hint #4). Grow the cultures with shaking at 37°C overnight.
2. Centrifuge each culture in three 500 ml centrifuge bottles in a Sorvall™ GSA rotor at 5,000 rpm (4000 X g) for 15 min at 4°C. Discard the supernatant and aspirate off the remaining liquid from the cell pellet (see Hint #5).
3. Resuspend the cells from one culture by vortexing in 150 ml of 50 mM EDTA in a single 500-ml centrifuge bottle.
4. Repeat Step #2 and aspirate off the supernatant (see Hint #6).
NOTE: Steps #5 through #27 apply to each 1-liter culture separately (volumes and manipulations are per liter). There is no advantage to pooling the two cultures until after Step #27.
5. Resuspend the cells in 40 ml of Buffered Glucose by vigorous shaking or vortexing.
6. Add 80 ml of 0.2 N NaOH/1% SDS. Mix by gentle inversion and place on ice for 15 min.
7. Add 60 ml of ice-cold Potassium Acetate Solution and mix by gentle inversion.
8. Centrifuge the lysed cell suspension in a Sorvall™ GSA rotor at 8,000 rpm (10,500 X g) for 15 min at 4°C (see Hint #7).
9. Filter the supernatant by decanting the solution into a beaker through 2 to 3 layers of cheesecloth.
10. Divide the filtered supernatant equally into four 50 ml round-bottom polypropylene "snap cap" centrifuge tubes (Nalge 3117-0500, and 3111-0030 closures). Snap the closures on the tubes and centrifuge in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 15 min at 4°C (see Hint #8).
11. To calculate the weight of the supernatants, pre-weigh three empty 250 ml centrifuge bottles. Then decant the supernatants into the bottles, dividing the supernatants equally into three 250 ml centrifuge bottles. Weigh the filled bottles and subtract the weight of the filled bottles from the empty ones to calculate the weight of the supernatants.
12. (Optional) To ensure that phage DNA is present in the supernatant, electrophorese a 10 μl sample of the supernatant (mixed with 2.5 μl of Lysis Mix) on a 0.8% Agarose/4X GBB minigel (see Hint #9). As a control, in a separate lane of the gel, electrophorese 200 ng of λ DNA digested with BstEII. Stain the gel with Ethidium Bromide to visualize the DNA (see Protocol ID#1192). You should see faint (approximately 10 ng) bands of DNA at 5 Kbp (the 9.2 Kbp covalently closed circular RF DNA molecule), 11 Kbp (nicked circular RF DNA) and 2 Kbp (9.2 Kb ssDNA). This will usually be superimposed on a smear of residual main chromosomal DNA, and there will be a heavy RNA band near the Bromphenol Blue dye front.
13. Add 150 ml of 95% (v/v) Ethanol to each bottle from Step #11. Allow the bottles to sit on ice for at least 30 min or store the precipitating DNA overnight to several days if convenient.
14. Pellet the DNA by centrifugation in a Sorvall™ GSA rotor at 8,000 rpm (10,500 X g) for 20 min at 4°C. Discard the supernatant and invert the tubes on paper towels for two minutes to drain residual supernatant. Wipe the inside wall of the centrifuge tubes with kimwipes.
15. Wash the pellets by adding 20 ml of 70% (v/v) Ethanol. Centrifuge as in Step #12 but only for 5 min. Discard the supernatant and invert the tubes on paper towels for two minutes to drain residual supernatant. Wipe the inside wall of the centrifuge tubes with kimwipes to remove remaining supernatant. Dry the pellets briefly (5 min) under vacuum.
16. Dissolve the DNA from the three pellets in a total volume of 10 ml TE. Transfer the DNA solution to an SS-34 centrifuge tube and centrifuge in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 15 min. Divide the supernatant equally into two 15 ml conical polypropylene centrifuge tubes.
17. Extract the supernatants with Neutralized Phenol using the double-spin method (see Hint #10) as follows: Centrifuge at 1,000 X g for 5 min to separate the phases and carefully remove the organic (lower) phase of the solution. Leave all of the interphase and aqueous (upper) phase in the tubes (see Hint #11). Centrifuge the tubes again to re-separate the phases. Carefully collect the aqueous phases into new 15-ml conical tubes, avoiding any interphase or organic solution.
18. Repeat the extraction with Phenol in Step #17.
19. Extract the supernatants from Step #18 with Chloroform using the double-spin method.
20. Pool the aqueous phases in a 50 ml snap-cap centrifuge tube or equivalent. Add TE to bring the net weight to 15 g. Add 7.5 ml of 7.5 M Ammonium Acetate and incubate on ice 15 min.
21. Centrifuge the tube in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 15 min to pellet large RNA species. Divide the supernatant equally into two centrifuge tubes.
22. Add 28 ml of 95% (v/v) Ethanol to each tube. Cover the tubes with the snap-caps, mix by inversion, and incubate for 15 min on ice (or overnight at 4°C) to precipitate the DNA.
23. Centrifuge the tubes in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 15 min to pellet the DNA. Discard the supernatant and invert the tubes on paper towels for two minutes to drain residual supernatant. Wipe the inside wall of the centrifuge tubes with kimwipes.
24. Wash the pellets by adding 20 ml of 70% (v/v) Ethanol. Centrifuge as in Step #23 but only for 5 min. Discard the supernatant and invert the tubes on paper towels for two minutes to drain residual supernatant. Wipe the inside wall of the centrifuge tubes with kimwipes to remove remaining supernatant. Dry the pellets briefly (5 min) under vacuum.
25. Dissolve each pellet in 4.8 ml of TE and pool the two DNA solutions in a single tube.
26. Centrifuge the tube in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 10 min. Pour the supernatant into a 15 ml conical tube. The DNA can be stored in this form at 4°C for months.
27. (Optional) Check for the presence of RF DNA as in Step #12.
NOTE: If the two preparations both have acceptably high yields and quality as determined by Step #27, the two preparations can be pooled.
28. Pre-weigh a 100 ml beaker and note the weight. Collect the DNA from Step #26 into the beaker and add sufficient TE to bring the net weight of the solution to 29 grams. Add exactly 30.90 g of CsCl, and dissolve the solid by carefully swirling the solution by hand.
29. Add exactly 2.54 grams of 10 mg/ml Ethidium Bromide (CAUTION! see Hint #1) and mix by swirling. You should now have 62.44 grams (39.77 ml) of a solution with a density of 1.57 g/ml (49.5% w/w CsCl) (see Hint #12).
30. Pour the solution through a 50 ml syringe barrel with a blunted 18 gauge needle as the funnel into a Beckman™ VTi50 tube. Seal the tube and centrifuge in the VTi50 rotor at 40,000 rpm (132,000 X g) at 20°C for at least 16 hr.
31. After centrifugation, there should be an intensely fluorescent pellet of RNA on the tube wall, sometimes accompanied by a purplish, non-fluorescent pellet of protein. The DNA band will often be invisible without UV illumination. Insert an 18-gauge needle into the tube near the top to act as an air inlet. Illuminating the solution with long-wave UV irradiation, collect the lower (RF) DNA band by carefully puncturing the tube from the side with an 18 gauge needle attached to a 5-ml syringe and gently drawing the solution into the syringe. Expel the collected DNA (typically 2 to 3 ml) into a 15-ml conical tube.
32. Extract the Ethidium Bromide from the DNA solution by adding an equal volume of Isopropanol, vortexing the solution gently, and discarding the upper (Isopropanol) phase after it separates fully from the lower phase. Repeat until the upper phase is completely colorless. Use a disposable polyethylene transfer pipette to draw off the upper phase.
33. After most of the upper phase from the last extraction has been removed, draw up the interphase into the pipette and allow the phases to separate within the stem of the pipette. The interphase is visible only as a refractive index discontinuity. Gently return the lower (aqueous) phase to the tube, being careful to avoid returning any of the upper (organic, Ethidium Bromide-rich) phase to the tube (see Hint #13).
34. Dilute the final aqueous phase to 10 ml with TE and transfer it to a clean polysulfone Oak Ridge tube or equivalent. Add 1 ml of 3 M Sodium Acetate and 22 ml of 100% (v/v) Ethanol. Mix by vortexing and allow the DNA to precipitate at 4°C for at least 1 hr.
35. Centrifuge the tube in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 15 min to pellet the DNA. Discard the supernatant and invert the tube on paper towels for two minutes to drain residual supernatant. Wipe the inside wall of the centrifuge tube with kimwipes.
36. Wash the pellet by adding 5 ml of 70% (v/v) Ethanol, being careful not to dislodge the pellet. Centrifuge as in Step #34 but only for 5 min. Discard the supernatant and invert the tube on paper towels for two minutes to drain residual supernatant. Wipe the inside wall of the centrifuge tube with kimwipes to remove remaining supernatant. Dry the pellet briefly (5 min) under vacuum.
37. Dissolve the DNA pellet in 5 ml of TE (see Hint #14).
38. Transfer the DNA solution to a conical 15-ml centrifuge tube. Extract once with Saturated Phenol and once with Chloroform, as in Steps #17 and #19.
39. Transfer the aqueous phase into a clean polysulfone Oak Ridge tube or equivalent. Add 500 μl of 3 M Sodium Acetate and 11 ml of 100% (v/v) Ethanol. Vortex to mix and allow the DNA to precipitate at 4°C for at least 1 hr.
40. Repeat Steps #35 and #36. Dissolve the DNA pellet in TE (500 μl per liter of culture) by vortexing; centrifuge tube briefly to drive the solution to the bottom of the tube and transfer the DNA solution to a 1.5 ml microcentrifuge tube. Microcentrifuge briefly to clear insoluble matter (if any) and transfer the supernatant to a new 1.5 ml microcentrifuge tube. Store the DNA at 4°C. The anticipated DNA concentration is approximately 300 μg/ml (see Hint #15).
41. Measure the absorbance (at 257 and 300 Angstroms) of a 1/10 dilution of the DNA solution. Calculate nucleic acid concentration by subtracting the reading at 257 Angstroms from the reading at 300 Angstroms and multiplying the result by 5 to derive the concentration of DNA in mg/ml (see Hint #16).
42. Electrophorese approximately 100 ng of DNA (diluted to 10 μl and mixed with 12.5 μl of lysis mix) on a 0.8% Agarose/4 X GBB gel to roughly calculate the concentration of RF DNA by comparing the bands of RF DNA to the DNA bands from the molecular weight markers. It should be noted in this context, however, that covalently closed circular DNA stains less intensely than linear double-stranded DNA.
Solutions
Potassium Acetate Solution
2 M Glacial Acetic Acid
Store at 4°C
3 M Potassium Acetate 
0.2 N NaOH/1% SDS
1% (w/v) SDS
0.2 M NaOH
Prepare fresh before use
Buffered Glucose
Autoclaving or filter sterilization is optional
25 mM Tris-Cl, pH 8
10 mM EDTA
50 mM Glucose
Store at 4°C
EDTA (250 mM stock)
Autoclave and store at room temperature
250 mM EDTA
Adjust pH to 8.0 with NaOH (CAUTION! See Hint #1)
Prepare in ddH2O
NZY Medium
For solid medium, add 20 g Bacto Agar (Difco) before autoclaving and pour into plates after autoclaving
Dissolve in 1 liter water
5 g NaCl
Store at room temperature
10 g NZ Amine A (Humko Sheffield Chemical)
Autoclave to sterilize
5 g Bacto Yeast Extract (Difco)
Adjust pH to 7.5 with NaOH (CAUTION! See Hint #1)
10 mg/ml Ethidium Bromide (CAUTION! See Hint #1)
10 mg/ml Ethidium Bromide
Prepared in TE
7.5 M Ammonium Acetate
Store at room temperature
Autoclave in tightly stoppered screw-cap bottle to prevent evaporation of the volatile salt
Dissolve 53 g Ammonium Acetate in 47 ml of ddH2O
Chloroform with Isoamyl Alcohol
Swirl to mix and store at 4°C
CAUTION! See Hint #1
Add 20.8 ml of Isoamyl Alcohol to 500 ml of Chloroform in the original bottle containing Chloroform.
Neutralized Phenol
Allow phases to separate and remove the aqueous (upper) phase
Equilibrate with Tris once more
Use water-saturated Phenol
Shake or vortex vigorously to equilibrate phases
Add one-tenth volume of 1 M Tris-HCl, pH 8.0
Use the lower phase as Neutralized Phenol
CAUTION! See Hint #1
TE
Autoclave and store at room temperature
pH adjusted to desired value
(unless otherwise noted, the pH is assumed to be 8.0)
1 mM EDTA
10 mM Tris-Cl
70% (v/v) Ethanol
95% (v/v) Ethanol
Lysis Mix (5X)
20 ml of Glycerol
18 ml of ddH2O
2 g SDS
2 ml of 40 X GBB
40 mg Bromphenol Blue
GBB (40X)
Dissolve in 500 ml ddH2O
Store at room temperature
Adjust final volume to 700 ml
45.94 g Anhydrous Sodium Acetate (or 76.16 g Trihydrate)
18.83 g Na2EDTA-2H2O
Adjust pH to 8.3 with Glacial Acetic Acid
142.4 g Tris Base
Lysis Mix
18 ml of ddH2O
2 g SDS
2 ml of 40X GBB
40 mg Bromphenol Blue
20 ml of 100% (v/v) Glycerol
For use, add 1 vol of Lysis Mix to 4 vol of electrophoresis sample
3 M Sodium Acetate
Store at room temperature
Adjust pH to 6 with Glacial Acetic Acid
3 M Sodium Acetate
Autoclave in a tightly-stoppered screw-cap bottle to prevent evaporation of Acetic Acid
Isopropanol
100% (v/v) Isopropanol
10 mg/ml Ethidium Bromide (CAUTION! See Hint #1)
10 mg/ml Ethidium Bromide (CAUTION! See Hint #1)
BioReagents and Chemicals
SDS
EDTA
Glucose
Tris-HCl, pH 8.0
Isoamyl Alcohol
NaOH
Chloroform
Ethanol
Phenol
Glycerol
Bromophenol Blue
Potassium Acetate
Ammonium Acetate
Sodium Chloride
Bacto Yeast Extract
Ethidium Bromide
Glacial Acetic Acid
NZ Amine A
Bacto Agar
Tris Base
Isopropanol
Sodium Acetate
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
2. fd-tet refers to the insertion of a Tn10 transposon into the genome of the fd bacteriophage. The transposon carries two genes that confer tetracycline resistance and its insertion prevents the normally high levels of replication of the phage DNA. The DNA from this bacteriophage strain and its derivatives are maintained at a low intracellular copy number, which reduces the loss of inserts that are toxic to the cell. The yields of RF DNA are higher from filamentous phage that lack the transposon insertion.
3. Be sure to propagate the vectors in an uninfectible strain (e.g., K802) and check for the accumulation of unwanted, infective pseudorevertants (see Protocol ID#2164).
4. The female bacterial strains, K802, or MC1061, used to propagate RF DNA plasmids, are resistant to infection because they lack pili projections. fd-tet-based vectors contain the Tn10 transposon, which confers tetracycline resistance and stability as a plasmid. fd-tet-based vectors are maintained at low copy number in host cells.
5. Do not discard the supernatant if you plan to isolate virions (see Protocol ID#2177).
6. Cell paste can be frozen indefinitely at 20°C at this point.
7. There will be a gelatinous pellet at the bottom of the bottle, and a stringy, gelatinous precipitate throughout the supernatant.
8. The supernatants resulting from this centrifugation step should be clear, often with a slight green tint. Any other tubes that can withstand the g-forces in this centrifugation step can also be used.
9. The 0.8% Agarose/4X GBB minigel is prepared as described in Protocol ID#1192. One volume of sample DNA is mixed with five volumes of Lysis Mix and electrophoresed as described.
10. The contributor advocates using the double-centrifugation method, as it increases the yield of the aqueous phase from each organic extraction.
11. The purpose of removing the organic phase is to lower the interphase into the narrow tip of the microcentrifuge tube so that the aqueous phase can be drawn off with high yield. Avoid removing the aqueous phase.
12. The author of this protocol notes that the RNA is not digested with RNase before loading on the Cesium Chloride/Ethidium Bromide gradient (as described in other DNA purification procedures using Cesium Chloride). By omitting the RNase step, the RNA molecular weight will remain high and the RNA will pellet on the wall of the centrifuge tube during centrifugation. A high concentration of Ethidium Bromide (639 μg/ml) is used because there is so much RNA present in the preparation. If, contrary to this precaution, the Ethidium Bromide is not added to excess, after centrifugation the solution will be a pale pink in color rather than the color of rose wine, and the DNA bands will be very low in the tube or even pelleted with the RNA.
13. Use a fresh transfer pipette for each extraction. Usually 3 extractions suffice; however, six extractions may be required if you do not use the trick of separating the phases in the stem of the pipette. The Isopropanol extractions will concentrate the solution, and sometimes CsCl will precipitate out. If this happens, simply add enough ddH2O until the salt redissolves.
14. The Ethanol precipitation should get rid of the bulk of the CsCl.
15. Because the RF DNA preparation is not digested with RNase and is purified by a single CsCl density gradient centrifugation, the DNA is usually contaminated with residual low molecular weight RNA molecules, which don't pellet in the CsCl density gradient. Usually these don't interfere with use of the RF DNA, but they do complicate quantitation by spectrophotometry. Therefore, the total nucleic acid concentration may be higher than the DNA concentration).
16. A good yield is 150 to 250 μg purified RF DNA per liter of original culture.
