Question

Once you had obtained a highly concentrated stock of purified phage, how would you separate the recombinant phage DNA away from the phage head and tail proteins?

Answer 1

Protocol for Phage DNA Extraction with Phenol:Chloroform

In this protocol you extract the genomic DNA from the phages in a lysate. The lysates
are “dirty” in that they contain spent media components, cell wall debris, flagella, nucleic
acids, bacterial proteins and unassembled phage proteins in addition to the phage
themselves. There are many methods for purifying phage DNA from the rest of the
lysate, This protocol is recommended specifically for phages.

for incubation times listed in each step.

Day 1: PEG Precipitation
1. Take 1 volume of phage high titer lysate (see below) and add PEG (10% PEG-
8000, 1 M NaCl final concentration) at a ratio of 1:2 precipitant:lysate. Mix gently
by inversion
a. As a rule of thumb, 1 ml of phage lysate with a titer of 1 x 1010 contains
about 0.5 μg of phage DNA, assuming the phage has a 50 kb genome
Typically 10 ml of a high-titer phage lysate is used for DNA extraction.
More or less lysate (up to 20 ml) may be used depending on the phage
stock titer and the expected genome size of the phage. For low-titer phage
stocks, up to 20 ml of lysate may be used. With high titer stocks of large-
genome phages, use 10 ml of lysate or less.
2. Incubate at 4°C overnight (this is ideal, can also do 60 minutes on ice if you need
to work faster). Most phages are stable like this for several days

Day 2: DNA Extraction
1. Centrifuge the PEG-precipitated sample at 10,000 xg for 30 minutes
2. Use 5mM MgSO4 to re-suspend the pellet, pipetting gently up and down. AVOID
introducing BUBBLES during the resuspension. Be sure to rinse down the sides
of the tube to obtain all of the pellet. Transfer to a labeled 2 mL epi tube
3. Using 500 µl of the concentrated sample: add 1.25 ul of DNaseI and RNase (20
mg/ml) and incubate at 37oC for 1hour.
a. (You can scale all volumes up if more than 500 uL are needed for
resuspension).
4. Add 1.25 µl of Proteinase K 20 mg/ml stock (20µg total) and 25 µl 10% SDS
stock (0.5% final concentration) and 20 ul of 0.5 M EDTA pH 8.0 (20 mM final).
Mix and incubate 1 hour at 60°C.
5. Allow to cool to room temperature.
6. Add an equal volume of phenol:chloroform (1:1) and invert several times.
7. Spin 3000 xg (6000 rpm on microfuge), 5 minutes room temperature.
8. Carefully transfer the supernatant with a wide-bore pipette tip to a fresh, labeled
2 mL epi tube.
9. Add an equal volume of phenol:choroform (1:1), invert.

10.Centrifuge as above and transfer the supernatant again.
11.Add an equal volume of chloroform, invert.
12.Centrifuge as above and transfer the supernatant to a fresh, labeled 2mL epi
tube.
a. Depending on volume here you may need to use a 15mL falcon tube
13.Add 1/10 volume of 3 M NaOAc (pH 7.5), and 2.5 volumes of ice cold ethanol
(100%). Mix well and incubate at -20°C overnight.
a. A fast method allows incubation on ice for 15-30 minutes.

Day: 3 DNA Precipitation
1. Centrifuge in a benchtop microfuge at max speed 20 minutes (10000 rpm for
15ml tubes as tube rating allows, 15000 rpm on the microfuge)
2. Carefully remove supernatant and fill tube halfway with 70% ethanol (made from
100% with purified nuclease free water), spin at max speed for 2 minutes
3. Repeat the above step one time (2nd 70% wash).
4. Remove as much ethanol as possible without disturbing the pellet – good idea to
hold onto the supernatant until DNA recovery has been confirmed
5. Leave tube open on bench ~ 15-30 minutes to let ethanol disperse
6. Dissolve in TE buffer

In this protocol use proteinase K, phenol:chloroform This solution use for denaturation of head and tail protein and separate DNA

Thank You!