Isolate and Purify Phage Genomic DNA
Grant Hartzog & Manny Ares, UC Santa Cruz Phage Genomics Lab
Required materials and equipment.
Phage buffer
37°C heatblock
80°C heatblock
Promega "Wizard" DNA purification kit, columns and syringes.
Phage precipitant solution
10 mM Tris pH 8.0, 0.1 mM EDTA (TE)
80% isopropanol
Low speed centrifuge and SA600 or SS-34 or equivalent rotor
clear plastic oak ridge tubes
eppendorf tubes
1ml aliquots of DNA resin from wizard kit, preheated to 37°C
1ml aliquot of sterile double distilled water, preheated to 80°C
Procedure
A. Harvesting Your Phage.
The goal of this part of the procedure is to obtain a large volume of highly
concentrated phage lysate that can be used for the DNA purification.
1. Perform a 10-plate infection and incubate for one day (allow to grow for two days
only if the phage you are working with requires it). If the plates have good "web patterns"
(i. e. the number of plaques is such that their area just covers the entire plate with only a
"web" of lawn remaining), they can be used to harvest the phage.
2. Flood the agar plates containing the plaques with phage buffer (PB) and let sit for
several hours (2-4 hr) at room temperature, or overnight at 4 °C.
3. Remove the PB (now containing phage) from the plate, avoiding any chunks of top
agar and place in a 50 ml conical falcon tube.
4. Centrifuge at 2500 x g (Usually this is around 3500-4000 rpm in a table top centrifuge)
for 20 minutes to pellet cell debris and top agar.
5. Transfer the supernatant (lysate) to a new 50 ml conical falcon tube, leaving the pellet
behind.
6. Screw the 50 ml conical tube containing the lysate to the steri-flip filter sterilization
unit.
7. Invert the steri-flip unit and using a mild vacuum, filter sterilize the lysate by passing it
through the steri-flip filter.
8. Remove the filter unit and replace it with the cap provided with the steri-flip using
aseptic technique.
9. Label the tube with your name, the date and the phage name, and store it at 4°C.
10. Estimate the titer of your lysate by spotting 5 ul of a 10 fold dilution series from 10-4
to 10-10.
B. Nuclease treatment of the lysate followed by NaCl-PEG precipitation of phage
particles.
The DNA purification kits have a limited capacity (20-40 ug), thus the amount of
lysate to be used for DNA purification will affect the performance of the resin
column used to purify it and the final DNA yield. Thus it is very important to start
with a known number of virus particles.
1. Empirically we have found that the following lysate volumes give a good phage DNA
yields without clogging the resin column:
a) For titers around 1x108 use 10 ml of lysate.
b) For titers around 1x109 use 5-6 ml of lysate.
c) For titers around 1x1010 use 4 ml of lysate.
d) For titers between 1x1011 and 1x1012 use 3 ml of lysate.
2. Transfer an appropriate amount (as determined above) of the filter-sterile phage lysate
into a clear 30 ml screw-capped polycarbonate Oak Ridge tube. Store the remaining
lysate at 4oC.
3. Put on gloves.
To degrade bacterial DNA and RNA in the lysate, you will use a mix of nucleases
(DNase I and RNase A). It is important to avoid contamination of your pipets,
workbench etc., with the nuclease mix. Discard the gloves after you use the
nucleases to avoid contamination of any material that you could touch with the
contaminated gloves. Note that the capsid of the phage protects its DNA from the
nucleases. However, if nucleases are present in later steps of the protocol, the
phage DNA may become degraded.
4. Add 4 microliters of nuclease mix per ml lysate to the Oak Ridge tube with the phage
lysate using your P-200 pipetman. For example if you used 4 ml lysate, add 16
microliters of nuclease mix, for 3 ml lysate, use 12 microliters of nuclease mix, etc..
5. Screw on the cap of the Oak Ridge tube and mix gently but thoroughly by swirling the
tube for several seconds. Don't vortex or otherwise introduce bubbles or excessive
shearing.
6. Incubate the lysate with nuclease mix in a 37°C water bath for 30 minutes.
7. Remove the Oak Ridge tube from 37°C and let sit undisturbed at room temperature for
1 hour.
C. Precipitate phage particles.
1. Using a 5-ml pipette, add 0.4 volume of phage precipitant solution to the nucleasetreated lysate. For example, if you used 10 ml of lysate to start, add 4 ml of phage
precipitant solution, if you used 4 ml to start, add 1.6 ml of phage precipitant solution,
etc.
2. Cap the tube and mix gently but thoroughly by swirling the tube for several seconds,
until you are sure that the phage precipitant solution is completely mixed with the lysate.
Look at the tube near a source of light so you can be sure that the solution is
homogeneously mixed. This mixing step is important for efficient precipitation. If you
see schlierin lines (visible streaks produced in a transparent medium as a result of
variations in the medium's density leading to variations in refractive index), then the
solutions are not well mixed.
3. Incubate tube on ice for 30 minutes.
NOTE: Longer precipitation times may give larger pellets that contain more
phage particles but these may also contain debris that can obstruct the resin
column. We recommend not precipitating the phage particles overnight.
4. Place the tube in a high-speed centrifuge (mark the side of the tube where you expect
to find the pellet) and spin at 10,000 x g for 20 minutes at 4°C.
In our Sorval centrifuge with the SA600 rotor, 8500 rpm is approximately 10,000
x g. NOTE: if you mark the outside edge of the cap of the tube with a sharpie, it
will be easier to find the phage pellet after centrifugation.
5. Put on gloves. Look for the pellet (your may want to draw a circle around it on the wall
of the tube). Pellets sometimes are transparent so do not discard the tube if you cannot
see a pellet! Decant the supernatant into the sink or a 50 ml conical falcon tube, being
careful not to disturb the pellet. Drain excess liquid from the pellet by inverting for 2 to 3
minutes on a paper towel.
D. Purify DNA from the phage particles.
1. Make 1 ml aliquots of the "DNA Clean Up Resin" suspension from the Wizard kit in 2
ml screw cap tubes and preheat in a 37°C heat block. Cutting the tip off of a 1 ml
pipetman (i.e. blue) tip is useful for distributing the resin suspension and associated
buffer. The buffer contains guanidinium thiocyanate, so handle it with care.
2. Warm 2-4 tubes of the "DNA Clean Up Resin" with 1 ml resin/buffer suspension each
in a 37°C water bath for several minutes.
3. Add 400 ul of TE to the phage pellet. For most phage there will be adequate NaCl in
the pellet to stabilize their capsids and this resuspension allows easy transfer to the
resin/buffer mixture.
If obtaining intact (i. e. undegraded DNA) appears to be a problem with your
particular phage it may be better to pipette the resin/buffer mixture directly onto
the pellet. It can then be more difficult to retrieve the resin for loading into the
column but may be necessary if the phage particle you are studying is fragile.
4. Resuspend the pellet by gently pipetting up and down, avoiding bubbles. It is not
necessary to completely redisolve the precipitate but it should be uniformly suspended.
5. To each of the two aliquots of resin/buffer suspension add 200 ul (or about half) of the
resuspended phage precipitate. If there is more volume in the pellet and you needed to
add more TE to get the pellet up, then use an additional aliquot of resin/buffer (and an
additional column, see below). Do not overload the 1 ml buffer aliquot with more than
200 ul of phage precipitate suspension, add more aliquots of buffer instead.
9. Invert the tubes with the resin-phage mixture several times until the solution is
homogeneous and let sit at room temperature.
To avoid DNA degradation, do not let the Phage-DNA-resin mixture to stand too
long at room temperature, do not store it at 4°C or place it at 37°C. Apply the
Phage-DNA-resin solution to the syringe-column set up immediately after it looks
homogeneous and continue with the 80% isopropanol wash and the elution step.
E. Isolate the phage genomic DNA.
1. Take a 3 ml syringe barrel (remove the plunger if present) and attach it onto a column
(both are supplied with the DNA purification kit). Set the column-syringe on top of a 1.5
ml microcentrifuge tube. The DNA-purification kit manual has a figure showing how the
column-syringe set up should look. Have a 3 ml syringe plunger ready to use (keep it in a
clean place. Set up several open 1.5 ml microcentrifuge tubes on a rack.
2. Prepare a new 1 ml cut-off blue pipette tip and transfer the resin-phage-genomic-DNA
solution to the column-syringe set up with the P-1000 pipettor. Use one column for each
ml of resin/buffer you have used.
Before transferring the resin, remember to mix it very well by inverting the tube
several times. Transfer 500-600 ul first and then the remaining 500-600 ul.
Pipette the resin up and down, before taking it.
3. Use the syringe plunger to push this solution through the column.
First attach the plunger carefully. Then start pushing slowly but firmly!
You will need to change the column set up to a new 1.5 ml microcentrifuge tube to have
enough space for the liquid that flows away from the column. Move to a new 1.5 ml
microcentrifuge tube as the waste comes through and keep pushing gently until the
plunger reaches the end of the column.
If you need to add extra-force to make the solution through, there is something
wrong. The column clogs when the phage capsid is not completely denatured and
even if you make the DNA-resin solution to go through using force, the isopropanol
wash will not work. At this point is better not to proceed with the protocol. Start the
purification over with changes. These are some suggestions:
a) The initial titer may be too high or the phage pellet obtained is too big. In
this case, reduce the starting volume of lysate used. Precipitate the phage for
a short time (30 min on ice).
b) Make sure to mix the phage pellet completely after the resin has been applied
to it. If it looks like there is too much material, split it to additional aliquots
of resin/buffer suspension and run through additional columns.
c) During the harvesting step, do not forget to remove the pellet cell debris from
the supernatant (lysate). Tranfer the supernatant (lysate) to a new tube
before filtering it.
d) Check that the sterile filtered lysate is still sterile. If the filter failed it is
possible bacteria have grown in the lysate during storage in the refrigerator.
If this happens filter again, and determine the titer again.
4. Wash salts and proteins off DNA using 80% isopropanol.
a. Remove the column from the syringe (unscrew it), and place the column onto a
clean 1.5 ml microcentrifuge tube.
b. Remove the plunger from the syringe and place the plunger on a paper towel.
c. Reattach the syringe (without the plunger) to the column and place the columnsyringe set up on a 1.5 ml microcentrifuge tube
d. Add 2 ml of 80% isopropanol to the column-syringe set up.
e. Carefully reattach the plunger to the column-syringe set up.
f. Gently (slowly) push the isopropanol through the column with the plunger. This
is a volume of 2 ml, so move the set up to another clean 1.5 ml microcentrifuge
tube as many times as the waste flows through.
g. Push the plunger all the way down and detach the column from the syringe.
h. Place the column containing your DNA on a new 1.5 ml microcentrifuge tube.
5. Dry the column and elute the DNA:
a. Make sure the heat block is on at 80°C and has ddH2O prewarmed for the elution.
b. Place the column in a clean 1.5 ml tube in a microcentrifuge and spin for 2
minutes at room temperature and maximum speed to remove the isopropanol. If
required, the lid of the microcentrifuge tube that holds the column can be removed
before placing it in the centrifuge.
c. Transfer the column to a clean, sterile, labeled 1.5 ml microcentrifuge tube with a
cap. This tube will contain your DNA.
d. Open the tube with the ddH2O, leaving it in the heating block at 80°C.
e. Take 50 ul of the 80°C ddH2O elution buffer up and down into the pipette tip to
warm it up. Then take 50 ul into the tip and immediately apply it to the column.
f. Wait 1 minute.
g. Centrifuge for 1 minute at room temperature at maximum speed. The ddH2O
elution buffer will go through the resin in the column and elute your purified
phage genomic DNA.
6. Store your DNA sample at 4°C until you are ready to quantify and analyze it.
F. Determine the concentration of your DNA.
DNA concentration can initially be determined using a spectrophotometer (or Nanodrop).
However you should also determine the concentration by gel analysis.
When we eluted with 50 ul TE, the DNA concentration was around 400 ng/ul in a final
volume of approximately 40 ul. For the restriction analysis we digested 1 ul DNA (400
ng). To measure the DNA concentration, further dilution of the DNA in TE was required.
Recipes
100 mM CaCl2 (100 mL)
Ingredient
CaCl2
ddH2O
Amount
1.47 g of CaCl2 ∙ 2H2O or
1.11 g of CaCl2 (anhydrous)
To 100 mL
Final Concentration
100 mM
To Prepare 1. Weigh out CaCl2 and place in a large beaker.
2. Add 90 mL of ddH2O and stir until the CaCl2 is dissolved.
3. Transfer the solution to a graduated cylinder, and add ddH2O to 100 mL.
To Sterilize Autoclave.
Store At room temperature; do not refrigerate.
Note: A good rule of thumb is that CaCl2 should be added to all solutions that come in
contact with phage, and, ideally, this should happen immediately before the solution is
used. The CaCl2 will precipitate out when cooling after autoclaving or when refrigerated.
Phage Buffer (PB; 1 L)
Ingredient
1 M Tris-Cl (pH 7.5)
1 M MgSO4
NaCl
ddH2O
100 mM CaCl2
Amount
10 mL
10 mL
4g
970 mL
10 mL
Final Concentration
10 mM
10 mM
68 mM
1 mM
To Prepare 1. Place all ingredients except CaCl2 into an Erlenmeyer flask.
2. Stir until the NaCl is completely dissolved.
3. Autoclave in the Erlenmeyer flask. Cool to 55°C in a 55°C water bath.
4. After cooling, aseptically add the CaCl2 and mix well by swirling.
5. Aliquot into sterile bottles, flasks, or tubes in 50-mL or 100-mL (or other) portions as
needed.
To Sterilize Autoclave.
Store At room temperature.
Notes 1. The CaCl2 should be added shortly before use. It will precipitate out after
autoclaving and cooling or when solutions are refrigerated.
Nuclease Mix
Ingredient
NaCl
ddH2O
DNase I
RNase A
Glycerol
Amount
0.088 g
4.25 mL
500 μL of 5-mg/mL stock
250 μL of 10-mg/mL stock
5 mL
Final Concentration
150 mM
0.25 mg/mL
0.25 mg/mL
50%
To Prepare 1. Dissolve the NaCl in 4.25 mL of ddH2O in a sterile 15-mL conical tube.
2. Add the RNase and DNase stock solutions, and mix with gentle inversion.
3. Add the glycerol slowly to a final volume of 10 mL, and mix with gentle inversion
until the solution is homogeneous.
4. Aliquot into microcentrifuge tubes.
To Sterilize Not required.
Store At –20°C (freezer).
Note: Be extra careful not to contaminate pipettes, benches, or other reagents with this
nuclease mixture, because it will degrade any DNA samples.
Phage Precipitation Solution
Ingredient
Polyethylene glycol 8000
(PEG 8000)
NaCl
ddH2O
Amount
30 g
Final Concentration
30%
19.3 g
To 100 mL
3.3 M
To Prepare 1. Add 19.3 g NaCl and 60 mL of ddH2O to a glass bottle with a screw cap,
and swirl until the NaCl is completely dissolved.
2. Slowly add 30 g of PEG 8000. Add only a few grams at a time, alternately swirling
and heating in a microwave until all the PEG is dissolved.
3. Add sterile water to 100 mL.
4. Add a sterile magnetic stir bar and stir until homogeneous.
5. Filter sterilize.
6. Dispense into sterile bottles or tubes.
To Sterilize Filter sterilize.
Store At room temperature.
Phage Precipitation Solution
Notes 1. The NaCl and the PEG 8000 are difficult to dissolve. Several iterations of
heating and stirring may be required. If there are precipitates in the solution, do not use it!
Rather, attempt to dissolve particulates by alternately heating the solution in a microwave
and swirling. Allow the solution to cool before using.