Using the Promega Wizard DNA Clean-Up Kit for DNA Extraction
OBJECTIVE
To extract a high yield, clean DNA sample from 1 mL of high titer phage lysate.
BACKGROUND
This is a quicker, easier, and safer alternative to PCI for extracting phage DNA using a commercial
DNA clean-up kit. In order to get a good yield you must have a high titer lysate with as little host
contaminants as possible (generally, a very clear lysate with no visible precipitates). Titers of 1010
work best, however the yield can be rather phage-dependent, as it is possible to get good yields
from titers of 109. Do not use lysates with titers greater than 1011 because the large amount of DNA
present in the sample will ‘clog’ the column and not elute properly.
APPROXIMATE TIME NEEDED
~ 1 hour with a 15 minute incubation period
MATERIALS NEEDED
Equipment
 Microcentrifuge
 Pipettors, micro- and serological
 95°C heating block
Consumables/Reagents (See online media preparation guides)
 Wizard Promega DNA Clean-Up Kit (Product #A7280)
 RNaseA (100mg/ml) and DNase I (Ambien, 2U/μL)
 80% isopropanol
 diH2O
 Microcentrifuge tubes
 3mL syringes (will only use the plunger)
 Kim Wipes
 15 mL conical tubes
 Appropriate micropipette tips
 Serological pipettes
HELPFUL TIPS

The clean-up resin provided in the Promega kit contains guanidinium thiocyanate,

which denatures protein. Handle with extreme care.
This procedure outlines the extraction for one phage, but more than one phage can be
done at the same time by multiplying everything in this protocol per phage.
PROCEDURES
1. Before starting:
a. Put the DNA cleantemperature to cool only before use- Use gloves when handling the resin.
b.
c. Make 15mL of 80% isopropanol fresh
2. From a high titer lysate, aliquot 1mL into a 15mL conical tube
3. Nuclease treat for host nucleic acid contaminants by adding 1μL RNaseA (100mg/ml) and 0.8μL
DNase I (Ambien, 2U/μL). Gently swirl to mix and let sit at room temperature for 15 minutes.
4. After 15 minutes add 2mL of resin from the kit and invert the tube several times to mix. Before
adding make sure that you have swirled all the precipitates back into solution in the resin. USE
GLOVES!
5. Set up two column bottoms and syringe tops. Add half of the DNA/resin mixture to each column.
(If you started with 1mL of lysate, adding 2mL of resin brings mixture to 3mL so you would add
1.5mL of the mixture to each column). Figure 1
Figure 1. How the initial setup should look. Two columns, with two syringe barrels per phage- each syringe
barrel will receive 1.5mL of the DNA/resin mixture which will be pushed through the columns, the DNA will
bind to the column, and the waste will be expelled into the microcentrifuge tubes for disposal. You will
continue working with these same two columns for the rest of the extraction.
6. For both columns: Using a plunger from a 3mL syringe, carefully push all the liquid through. This
may be difficult if your sample is concentrated. It is really important to maintain even pressure
and not let the plunger buck back out. When you finish expelling the liquid, dry to the last drop
by touching the column to a Kim Wipe-then unscrew the column. ONLY THEN can you let go of
the plunger.
7. Remove the plunger from the syringe barrel, and then reattach the syringe barrel to the column.
8. Wash both columns by slowly and carefully adding 2mL 80% isopropanol to each. Use the barrel
(you can use the same one) to force the liquid through. Again you may encounter resistance, but
don’t let the plunger buck back out. Maintain even pressure and dry to the last drop as in step 6.
Unscrew the column.
9. Repeat steps 7 and 8 twice more for a total of three washes. You can dispose of the tubes
containing the waste from each wash, but do not dispose of the column!
10. Put each of the columns in new 1.5mL microcentrifuge tubes. Spin at top speed (13,00014,000rpm) for 5 minutes to remove residual isopropanol. (You can use your two columns to
balance each other.)
11. Take the columns out of the 1.5mL tubes and put them on an
-
1 minute.
12. Put the columns in new microcentrifuge tubes. Apply 50μ
each column. (Leave the water on the heat block to keep it hot).
13. Incubate for 1 minute.
14. Spin both at top speed (13,000-14,000rpm) for 1 minute.
15. Repeat steps 12-14 twice more to complete three elutions per column. Keep these tubes from
each elution- they contain the DNA!
16. Test DNA in nanodrop and by agarose gel. Save DNA by freezing at -
-