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Isolation of Crude RF DNA from Small 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 "mini" preparations of infected bacterial cells. The RF DNA is propagated as an fd-tet-based vector (see Hint #2). The procedure is similar to the alkaline lysis protocols for standard isolation of plasmid DNA from bacteria (see Protocol ID#9033); 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 a 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. Pour the saturated culture into a 1.5 ml microcentrifuge tube and microcentrifuge at top speed for 30 sec to pellet cells.
3. Aspirate the supernatant, re-centrifuge the tube for 10 sec, and remove the remaining supernatant.
4. Resuspend the cells in 100 μl of Buffered Glucose by vortexing vigorously.
5. Add 200 μl of 0.2 N NaOH, 1% SDS and mix thoroughly but gently by inversion (do not shake or vortex). Incubate the tube on ice 10 min.
6. Add 150 μl of ice-cold Potassium Acetate Solution and mix thoroughly but gently by inversion (don't shake or vortex). Incubate the tube on ice 10 min.
7. Centrifuge the tube for 10 min at maximum speed in a microcentrifuge.
8. Carefully pipette the supernatant to a new 1.5 ml microcentrifuge tube. If the supernatant is not clear, repeat Steps #7 and #8.
9. To the supernatant, add 900 μl of 95% (v/v) Ethanol and vortex or invert to mix. Centrifuge the tube for 10 min at maximum speed in a microcentrifuge.
10. Aspirate the supernatant, re-centrifuge the tube for 10 sec, and remove the remaining supernatant. Dry the pellet briefly (5 min) under vacuum.
11. Dissolve the pellet in 200 μl of TE.
12. Add 100 μl of 7.5 M Ammonium Acetate and mix by vortexing. Incubate the tube on ice for 10 min.
13. Centrifuge the tube for 10 min at maximum speed in a microcentrifuge (see Hint #3).
14. Transfer the supernatant (containing the RF DNA) to a new 1.5 ml microcentrifuge tube, being careful to avoid the pellet.
15. Add 600 μl of 95% (v/v) Ethanol to the supernatant, vortex to mix, and centrifuge for 10 min at maximum speed in a microcentrifuge.
16. Aspirate the supernatant, re-centrifuge the tube for 10 sec, and remove the remaining supernatant. Dry the pellet briefly (5 min) under vacuum.
17. Dissolve the pellet of RF DNA in 500 μl of TE, pH 7.2.
18. Add 1 μl of 10 mg/ml RNaseA and carry out the digestion at 37°C for 15 min.
19. Extract the supernatant with Phenol/Chloroform 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.
20. Repeat the extraction with Neutralized Phenol in Step #19.
21. Extract the supernatant from Step #20 with Chloroform using the double-spin method.
22. Transfer up to 350 μl of the final aqueous solution to a fresh 1.5 ml microcentrifuge tube and add 35 μl of 3 M Sodium Acetate and 770 μl of 100% (v/v) Ethanol. Allow the DNA to precipitate at 4°C for at least 30 min.
23. Centrifuge the tube for 15 to 30 min at maximum speed in a microcentrifuge. Aspirate the supernatant and wash the pellet with the addition of 1 ml of 70% Ethanol.
24. Centrifuge the tube for 5 min at maximum speed in a microcentrifuge. Aspirate the supernatant, then re-centrifuge for 10 sec. Remove remaining supernatant from the DNA pellet.
25. Dissolve the pellet in 50 μl of TE. 5 μl of the DNA solution contains approximately 50 ng RF DNA that can be visualized by electrophoresis and staining of an Agarose gel (see Hint #2; and see Protocol ID#2170 for instructions on running the agarose gel).
Solutions
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)
1 mM EDTA
10 mM Tris-Cl
70% (v/v) Ethanol
Phenol/Chloroform
Allow phases to separate and remove the aqueous (upper) phase
Use the lower phase as Phenol/Chloroform
Equilibrate with Tris once more
Store at 4°C
Add one-tenth volume of 1 M Tris-HCl, pH 8.0
Mix an equal volume of Neutralized Phenol with Chloroform
Shake or vortex vigorously to equilibrate phases
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.
95% (v/v) Ethanol
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
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)
Dissolve in 1 liter water
5 g NaCl
For solid medium, add 20 g Bacto Agar (Difco) before autoclaving and pour into plates after autoclaving
Store at room temperature
10 g NZ Amine A (Humko Sheffield Chemical)
Autoclave to sterilize
5 g Bacto Yeast Extract (Difco)
BioReagents and Chemicals
NZ Amine A
Tetracycline
Ethanol
Bacto Yeast Extract
RNaseA
Glacial Acetic Acid
Glycerol
Isoamyl Alcohol
Chloroform
Phenol
Potassium Acetate
Tris-Cl
Ammonium Acetate
NaOH
SDS
Sodium Chloride
Glucose
EDTA
Sodium Citrate
Protocol Hints
1. CAUTION! This substance is a biohazard. Please consult this agent's MSDS for proper handling instructions.
2. This protocol is designed for fd-tet-based vectors. 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. The yields of RF DNA are higher from filamentous phage that lack the transposon insertion.
3. High molecular weight RNA is presumably precipitated and pelleted in this step.
4. The contributor advocates using the double-centrifugation method 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.
