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fd-ted-based VECTORS
Isolation of Crude Replicative Form (RF) DNA from Mid-Size Cultures of Bacteria Harboring fd-ted-based Vectors
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
This protocol describes the isolation of Replicative Form (RF) bacteriophage DNA from "midi" preparations of infected bacterial cells (40 ml cultures). The RF DNA is propagated as an fd-tet-based vector (see Hint #2). The procedure is similar to the standard alkaline lysis protocol for isolation of plasmid DNA from bacteria (see Protocol ID#574); however, this protocol includes additional purification steps to isolate the bacteriophage RF DNA. The resulting DNA preparation is crude compared to the large-scale preparation that employs Cesium Chloride gradient centrifugation for purification.
Procedure
1. Inoculate a 1.7 ml NZY culture containing 20 μg/ml Tetracycline in a 13 ml plastic culture tube with a colony of fd-tet bacteriophage-infected bacteria. Incubate the tube vertically with shaking at 300 rpm overnight at 37°C.
2. Inoculate a 40 ml NZY culture containing 20 μg/ml Tetracycline in a 250 ml culture flask with 40μl of the culture from Step #1. Incubate the flask with shaking at 300 rpm overnight at 37°C.
3. Pour the culture into a 50 ml round-bottom polypropylene "snap cap" centrifuge tube (Nalge 3117-0500, and 3111-0030 closures) (see Hint #3). Snap the closure on the tube and centrifuge in a Sorvall™ SS-34 rotor at 5,000 rpm (3,000 X g) for 10 min at 4°C.
4. Aspirate the supernatant, re-centrifuge the tube for 1 min, and remove the remaining supernatant. The cells can be placed at -80°C for long-term storage.
5. Resuspend the cell pellet in 3 ml of Buffered Glucose.
6. Add 6 ml of 0.2 N NaOH/1% SDS and mix gently by inversion. Incubate on ice for 10 min.
7. Add 4.5 ml of ice-cold Potassium Acetate Solution and mix gently by inversion.
8. Centrifuge the tube in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 10 min.
9. Filter the supernatant by decanting the solution through 2 to 3 layers of cheesecloth into a new centrifuge tube.
10. Centrifuge the tube in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 10 min.
11. Transfer the supernatant to a new centrifuge. Add 33 ml of 95% (v/v) Ethanol, snap the closure on the tube and incubate on ice for at least 20 min.
12. Centrifuge the tube in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 20 min.
13. Aspirate the supernatant, re-centrifuge the tube for 1 min, and remove the remaining supernatant.
14. 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.
15. Dissolve the pellet in 4 ml of TE.
16. Add 2 ml of 7.5 M Ammonium Acetate Solution, mix by vortexing, and incubate on ice for at least 15 min.
17. Centrifuge the tube in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 10 min at 4°C. Transfer the supernatant to a new tube.
18. Add 15 ml of 95% (v/v) Ethanol and incubate the tube on ice for at least 20 min.
19. Centrifuge the tube in a Sorvall™ SS-34 rotor at 15,000 rpm (27,000 X g) for 20 min at 4°C.
20. Aspirate the supernatant, re-centrifuge the tube for 1 min, and remove the remaining supernatant.
21. Wash the pellets by adding 20 ml of 70% (v/v) Ethanol. Centrifuge as in Step #19 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.
22. Dissolve the pellet in 500 μl of TE, pH 7.2 and transfer the DNA solution to a 1.5-ml microcentrifuge tube.
23. Add 1 μl of 10 mg/ml RNase A. Mix briefly by vortexing and incubate at 37°C for 15 min.
24. Extract the supernatant with Saturated Phenol using the double-spin method (see Hint #4) as follows: Centrifuge at maximum speed in a microcentrifuge 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 tube (see Hint #5). Centrifuge the tube again to re-separate the phases. Carefully collect the aqueous phase into a new tube, avoiding any interphase or organic solution.
25. Extract the supernatant from Step #24 with Chloroform using the double-spin method and transfer the aqueous phase to a new microcentrifuge tube.
26. Add 40 μl of 3 M Sodium Acetate and 1 ml of 95% (v/v) Ethanol. Incubate on ice for at least 20 min.
27. Centrifuge the tube at maximum speed in a microcentrifuge for 30 min.
28. Aspirate the supernatant, re-centrifuge the tube for 10 sec, and remove the remaining supernatant.
29. Wash the pellets by adding 500 μl 70% (v/v) Ethanol. Centrifuge as in Step #27 but only for 5 min. Discard the supernatant, re-centrifuge for 10 sec, and remove the remaining supernatant. Dry the pellet briefly (5 min) under vacuum.
30. Dissolve the DNA pellet in 50 μl TE. The DNA solution can be stored long-term at 4°C or at -80°C. RF DNA is typically at a concentration of between 50 and 200 μg/ml and can be visualized by electrophoresis and staining of an Agarose gel (see Hints #2 and Hint #6; and see Protocol ID#2170 for instructions on performing agarose gel electrophoresis).
Solutions
95% (v/v) Ethanol
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
Tetracycline (1000X)
Mix thoroughly and store at 20°C in a tube covered with aluminum foil
Filter Sterilize
40 ml of 40 mg/ml Tetracycline
Add 40 ml of autoclaved 100%(v/v) Glycerol
NZY Medium
Adjust pH to 7.5 with NaOH (CAUTION! see Hint #1)
5 g NaCl
For solid medium, add 20 g Bacto Agar (Difco) before autoclaving and pour into plates after autoclaving
Dissolve in 1 liter water
Store at room temperature
10 g NZ Amine A (Humko Sheffield Chemical)
Autoclave to sterilize
5 g Bacto Yeast Extract (Difco)
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
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
10 mg/ml RNaseA
Thaw and refreeze as needed
Store at -20°C
Dissolve RNaseA at 10 mg/ml in
Dispense 200 μl portions into 500 μl microcentrifuge tubes
Heat at 95°C for 3 min
0.1 M NaCl
25 mM Sodium Citrate, pH 5.5
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
TE
Autoclave and store at room temperature
pH adjusted to desired value
(unless otherwise noted, the pH is assumed to be 8.0)
10 mM Tris-Cl
1 mM EDTA
70% (v/v) Ethanol
BioReagents and Chemicals
Ethanol
NZ Amine A
Tetracycline
Bacto Agar
Isoamyl Alcohol
Chloroform
Phenol
Glycerol
Potassium Acetate
Ammonium Acetate
Tris-Cl
Glacial Acetic Acid
RNaseA
Bacto Yeast Extract
NaOH
Sodium Acetate
EDTA
SDS
Sodium Chloride
Sodium Citrate
Glucose
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. Other tubes designed for the SS-34 rotor may also be used.
4. The contributor advocates using the double-centrifugation method so as to increase the yield of the aqueous phase from each organic extraction.
5. 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 a high yield. Avoid removing the aqueous phase.
6. There will be roughly equivalent amounts of nicked circular RF DNA and residual host genomic DNA, and a heavy single-stranded phage DNA.
