Phagehunting Program
Using the Promega Clean-Up Kit for DNA Isolation
OBJECTIVE
Extract phage genomic DNA using the Promega DNA Clean-Up Kit.
BACKGROUND
There are various ways to yield clean DNA from a high titer phage lysate. This one requires about
12 mL of sample, and utilizes procedures that involve a condensed version of DNA precipitation
and as well as a commercial DNA clean-up kit.
APPROXIMATE TIME NEEDED
Reagent Preparation: ~1 hour
DNA Degradation and Precipitation: ~2.5 hours Incubation + 30 minutes active
DNA Isolation + Elution: ~30 minutes
MATERIALS NEEDED
Equipment
 Centrifuges, high-speed and micro Stir plates and stir bars
 Microwave
 37°C water bath
 80°C heating block or water bath
 Pipettors, micro- and serological
Consumables/Reagents (See online media preparation guides)
 Serological pipettes
 80% isopropyl alcohol
 Phage precipitant solution (PEG 8000, dH2O, NaCl)
 Nuclease Mix (NaCl, dH2O, DNase I, RNase A, 100% glycerol)
 Promega DNA Clean-Up Kit (Product #A7280)
 Sterile microcentrifuge tubes
 15 mL conical tubes
 3 mL or 5 mL syringe
 Appropriate micropipette tips
HELPFUL TIPS

The clean-up resin provided in the Promega kit contains guanidinium thiocyanate,
which denatures protein. Handle with extreme care.
PROCEDURES
Reagent Preparation
1.
Prepare 100 mL of phage precipitation solution.
a. Obtain 19.3 g of NaCl, dissolve it in 60 mL of dH2O in a 250 mL Erlenmeyer flask for a final
concentration of 3.3 M. Swirl until the NaCl is completely dissolve. This may take up to 5
minutes. Do not use a stir plate and stir bar.
b. Slowly add 30 g of PEG 8000 for a final concentration of 30%. Add a few grams at a time,
alternating between swirling and heating using the microwave for about 15 seconds at a
time. Avoid boiling the solution. Continue adding until all PEG is dissolved. This may take
up to 20 minutes, and the final solution should look cloudy.
c. Add sterile water to a final volume of 100 mL. The amount to be added is generally
around 13 mL, but be sure to adjust accordingly.
d. Add a sterile magnetic stir bar and stir the solution on a stir plate until homogeneous.
This generally takes about 30 minutes, and the final solution must be water clear. In the
mean time, proceed to step #2 to prepare the nuclease mix.
e. Filter sterilize the solution into 50 mL conical tubes. This takes a while (up to 10 minutes
per 50 mL conical tube) as the PEG solution is very viscous.
f.
2.
Store at room temperature. This solution cannot be used while warm or hot, or if there
are still precipitates in the solution.
Prepare 10 mL of nuclease mix.
a. Obtain 0.088 g of NaCl, dissolve in 5.00 mL of dH2O in a sterile 15 mL conical tube, to a
final concentration of 150 mM.
b. Add DNase I to a final concentration of 10 U/mL. For example, if the stock solution is
2000 U/mL, add 20 μL. Mix by gentle inversion. DO NOT VORTEX.
c. Add RNase A to a final concentration of 0.25 mg/mL. For example, if the stock solution is
100 mg/mL, add 25 μL. Mix by gentle inversion. DO NOT VORTEX. Do not confuse
between RNase I and RNase A, the latter is appropriate for this nuclease mix, and is
generally stored at room temperature. A common source in research laboratories for
RNase A are from MiniPrep kits.
d. Add 5.00 mL of 100% glycerol to a final concentration of 50%. Mix with gently inversion
until the solution is homogenous. DO NOT VORTEX.
e. Aliquot into microcentrifuge tubes (DO NOT USE DIRECTLY FROM 15 mL CONICAL
TUBE), and store at –20°C.
Bacterial DNA Degradation
3.
Transfer 10 mL of filtered, pure lysate into a 15 mL conical tube. Store the remaining lysate at
4°C; do not deplete the lysate stock.
4.
Put on gloves, then add 40 μL of the prepared nuclease mix. Be extremely careful not to
contaminate the sample, pipettes, or the nuclease mix solution. Mix by gently inversion. DO
NOT VORTEX.
5.
Incubate at 37°C for 30 minutes. A water bath generally works well: tighten the cap and
simply allow the tube to float in the water bath. Heat blocks are generally undesirable as they
do not envelop the entire tube.
6.
After 30 minutes have elapsed, let the tube sit undisturbed at room temperature for 1 hour.
Precipitate Phage Particles
7.
Put on a new pair of gloves. Using a 5 mL serological pipette, add 4 mL of the prepared
phage precipitant solution to the nuclease-treated lysate.
8.
Mix by thorough but gentle inversion.
9.
Incubate 30 minutes on ice. For better results, leave at 4°C overnight.
10.
Place the tube in a high-speed centrifuge, spin at 10,000 RPM for 20 minutes. Mark where the
pellet is expected.
11.
Decant the supernatant, but be careful not to disturb the pellet. Drain excess liquid from the
pellet by inverting and letting sit for 2 to 3 minutes on a paper towel.
Isolate Phage Genomic DNA
12.
Put on a new pair of gloves. Add 1 mL of pre-warmed (37°C) DNA Clean-Up Resin from the
Promega kit to the precipitated phage pellet. PEG-containing precipitate may also be in the
tube, hence pay attention to where the DNA pellet is expected and try not to “re-suspend”
precipitate or pellet that were not expected.
13.
Re-suspend the pellet by gently pipetting up and down. Gently swirl to mix.
14.
Allow DNA to bind to the column.
a. Attach the column supplied with the kit to the syringe.
b. Transfer ~1 mL of resin-phage-genomic DNA solution to the column. Note: If the resinphage-genomic DNA mixture looks viscous or contains jelly-like precipitate, the PEGcontaining precipitate, instead of the DNA-containing precipitate, was likely resuspended. Repeat steps #10 and #11.
c. Using the syringe plunger, push the solution through the column.
15.
Wash salts and proteins off DNA.
a. Disassemble the column, plunger, and syringe.
b. Re-attach the syringe to the column. Add 2 mL of 80% isopropyl alcohol to the column.
c. Push the isopropanol through the column using the plunger.
16.
Dry the column.
a. Remove the column. Centrifuge it for 5 minutes at maximum speed to remove the
isopropanol. Use a clean microcentrifuge tube with its lid removed. Make sure the
column and is securely fastened (“clicked”) to the top of the microcentrifuge tube.
b. Remove the column, decant any eluted liquid, and then reassemble the column to the
microcentrifuge tube.
c. Centrifuge for 1 minute at maximum speed to remove the residual alcohol.
DNA Elution
17.
Transfer the column to a clean, sterile, labeled microcentrifuge tube.
18.
Rapidly apply 50 μL of pre-warmed (80°C) sterile water to the resin in the column.
19.
Centrifuge for 1 minute to elute purified phage genomic DNA.
20.
The purified DNA sample may be stored at 4°C temporarily, or at –20°C for a few days. It is
recommended that samples be placed at –80°C for long-term storage.
21.
Measure the concentration of DNA on the NanoDrop. (See TOOLBOX: Measuring DNA
Concentration).