Characterization and sequencing of Pseudoaltermonas spp. phage from Eel Pond
(aka Fun with Phage)
Alison Ling
University of Colorado
Summer 2010 @ MBL
Abstract:
Two different phage were isolated from closely related Pseudoaltermonas spp. strains
from Eel Pond. The phage appear icosahedral as visualized by TEM, and at least one has a tail.
One phage may be lysogenic as evidenced by unexpected lack of infection in several assays.
Adsorption was characterized according to first-order kinetics. Different multiplicity of infection
is found to result in higher phage titers at lower MOI, due to prolonged infection before complete
cell lysis. Phage DNA was extracted using polyethylene glycol precipitation, CsCl gradient
ultracentrifugation, and phenol-chloroform extraction. 22 high-quality sequences were obtained,
which are similar by BLASTX to host genes (7), phage genes (4), other bacterial genes (5), and
no hits (6). Sequences with no hits may be novel phage genome sequences, but more sequence
coverage is needed before definitive conclusions can be drawn. The induction of lysogenic
phage was achieved from 2 of 8 marine isolates treated with mitomycin C, and resulting phage
particles were visualized as tailed phage by TEM.
Introduction:
Viral abundance and diversity in the environment is vast. Marine surface water is
estimated to contain between 10 and 100 times more viral particles than bacterial and archaeal
cells1. Viruses in seawater, primarily bacteriophage, play a major role in biogeochemical
cycling, population dynamics, genetic transfer, and biomass buoyancy1,2. It is estimated that
about a quarter of the organic carbon in the ocean is released through the viral shunt each day3.
Additionally, viral diversity is largely uncharacterized. 60-80% of viral sequences recovered
from the environment have no apparent homology to any sequences in public databses1,4.
Because viruses do not have universally conserved sequences as do the three domains of life,
primers cannot be designed to target all of viral diversity. Although phage dynamics, genomics,
and environmental effects are now known to drive ecosystem processes, they remain poorly
understood.
The idea of applying phage to control populations of harmful bacteria in a medical
context has been around since the early 20th century, but the explosion of antibiotics displaced its
significance in the medical field for a time, and a dearth of understanding regarding their
dynamics further hindered its practical application. Targeted bacteria can develop resistance to
phage through random mutation, necessitating the use of a ‘cocktail’ of phages to work
effectively while evading resistance5. Phage genomes can also be trawled for potential antibacterial molecules6. While phage therapy in medical fields will probably remain controversial,
the application of phage therapy to environments of engineering relevance is a promising avenue
for ameliorating microbially induced corrosion.
My goals in undertaking this study are to gain an understanding of phage processes
through isolation, characterization, and sequencing that I can use in the future to design phage
therapy for microbially induced corrosion in engineered systems. These systems are subject to
degradation by acidification and oxidation by microbial biofilms. Recent work to characterize
the diffusion of phage in biofilms has found that biofilms can act as a reservoir for phage
diversity7. It is very likely that phage already play a role in controlling biofilm, and if they can
be isolated and characterized, they could be used to control corrosion. Additionally, one study
engineered a phage to produce an enzyme that degrades the polysaccharide structure and resulted
in 100x more effective biofilm disruption and cell kill rates8. While therapy using engineered
phages cannot be legally applied to the field, its further development could eventually lead to
changes in legislation.
In this project, I isolate a phage from a marine environment and conduct
cultureexperiments and molecular work to characterize it. Lytic phage infection and cell growth
occurs in several different steps: adsorption and attachment to cell, injection of nucleic acid,
eclipse period of biosynthesis, phage accumulation, and lysis. General knowledge in the
virology field dictates that a given virus has an ideal multiplicity of infection (MOI) that results
in the highest ratio of infective to non-infective particles9, but in-depth characterization of MOI
effects in phage has not yet been undertaken. The optimized production of infectious particles
will be important in developing phage for phage therapy treatments.
Phage infections fall under three major categories: lytic, in which the host cell is lysed to
create more phage; chronic, in which the cell produces phage particles without lysing; and
lysogenic, in which the phage genome is inert while integrated into the host genome as a
prophage. Lysogeny has been found to be inducible by the mutagen mitomycin C in about 40%
of marine isolates10. The choice to enter lysogenic or lytic infection in a host is determined by
the concentration of phage inside the cell and cell size11, but is still poorly understood. A better
grasp of the processes involved in lysogeny will be necessary if the genomes of treatment phages
are to be maintained over time without the need for re-application.
In this study, I characterize adsorption rate and attempt to describe the growth rate and
burst size of the isolated phage. I also infect cells at different MOI and attempt to compare the
abundance of phage produced and the ratio of infective to non-infective particles. As a sideproject, I use mitomycin C to observe lytic induction of prophage in marine bacterial isolates.
In the interest of time and the absence of a pressing need for publishable results,
experimental replicates were not performed for most procedures, and failed experiments were
not repeated more than three times.
Methods:
Phage Enumeration:
Infective viral particles were measured using plaque assays in 100mm round plates and in
24-well tissue culture plates. For round plates, 500 uL of 0.4 OD600 cells were combined with
500 uL of phage innoculum and incubated for 20 minutes. 500 uL of infected cells were added
to 3 mL of SWC soft agar (0.5% noble agar) and the mixture was spread on 100 mm SWC agar
plates. Plates were incubated at room temperature overnight, and checked for plaques in the
morning.
The use of 24-well plates enabled the measurement of samples of unknown titer without
using a huge number of large plates. Viral dilutions were performed in 96-well deep plates () to
-4 or -6 log dilutions depending on the assay. 50 uL of 1.0 OD600 cells and 10 uL viral dilution
were incubated together for 15 minutes to allow adsorption. 940 uL of soft agar (0.5%) at 42°C
was added to each well and the mixture was immediately transferred to a well in a 24-well
culture plate.
Total viral particles were enumerated using SYBR green staining and epifluorescence
microscopy according to Patel et al. (2007) using 0.8 um nitrocellulose membrane support filters
(Millipore) and 0.02 um Anodisc filters (Whatman)12. Optical density of cultures were measured
using a Genesys 20 spectrophotomoter (ThermiScientific).
Phage isolation:
Ten isolates from Eel Pond grown on SWC agar, including 6 lumos, were screened for
phage susceptibility. Eel Pond water was filtered through a 0.2 um polycarbonate syringe filter
to yield cell-free phage innoculum. This solution was used as phage innoculum in a 100mm
plaque assay. Lytic viral infection occurred in two phage cultures (A and C), both related to
Pseudoaltermonas species. Plaques were picked and suspended in 1 mL of sterile water, which
was filtered to remove cell debris. Viral lysate stock was stored at 4°C.
Host identification:
Well-isolated colonies from host cultures A and C were picked and boiled at 95C for 10
minutes in a thermocycler (). in 10 uL sterile DNA-free water for 20 minutes. The resulting cell
lysate was amplified using universal bacterial 16S primers 8F/1492R. The PCR reaction
includes 10 uL sterile water, 12.5 uL Invitrogen 2X Master Mix, 2 uL of each primer, and 1 uL
of template DNA per reaction. DNA was denatured at 95°C for 5 minutes followed by thirty
cycles of 95°C for 30 second, 46°C for 30 seconds, and 72°C for 1:30. The final elongation step
occurs at 72°C for 5 minutes.
Phage DNA and sequence preparation:
Phage DNA was extracted from 100 mL of phage lysate using polyethlyne glycol, CsCl
gradient ultracentrifugation, and phenol-chloroform extraction (See Appendix A for full
protocol). Polyethylene glycol and cesium chloride gradient procedures were modified from
from Thurber et al (2009)13 and phenol-chloroform extraction was modified from Current
Protocols in Molecular Biology14. Alternate extraction methods without CsCl gradient
purification were also performed using CTAB surfactant plus phenol-chloroform and phenolchloroform extraction alone.
Resulting DNA was checked for bacterial contamination using 8F/1492R primers as
described previously. Previous phage sequencing projects have used six-cutter restriction
enzymes such as HindIII or HaeIII to digest extracted DNA into clonable fragments15. Here,
DNA aliquots with the least 16S contamination was digested with six-cutter BanI (New England
Biosciences) for one hour according to Molecular Cloning16. Digests were phenol-chlorofom
extracted and precipitated using sodium acetate and ethanol. DNA was adenilated at 65°C for 20
minutes using 50% Invitrogen PCR 2X Master Mix, cloned into TOPO vector (Invitrogen), and
transformed into electrocompetent E. coli. Clones were picked and sequenced using 96-well
Sanger capillary sequencing.
Sequences with poor quality or a high number of N’s were discarded. 25 of the 38
sequences obtained had 100% similarity to the pZero++ Amp cloning vector used in the TOPO
kit, so Phrap’s cross-match program was used to trim vector from the sequences. The remaining
28 sequences were compared to BLAST databases.
Phage Growth Parameters:
Phage adsorption rate and burst size were assayed according to Hyman et al. (2009)17.
Briefly, host cultures were infected at an MOI of approximately 1. In the adsorption assay,
phage was added to cells and cultures were incubated at room temperature. Samples were
collected every minute for ten minutes. Cells were spun down, and free phage in supernatant
was collected and assayed for PFUs in 24-well plates.
A one-step growth curve was conducted to determine growth rate and burst size
according to Hyman et al. (2009)17. Phage were added to exponentially growing cells and
allowed to adsorb at room temperature for 15 minutes. Infected cells were then pelleted by
centrifugation for 5 minutes at 8000 x g. Cells were washed of free virus, re-suspended in fresh
media, and incubated at 37°C. Samples were collected every 10 minutes for 100 minutes.
Samples were treated with chloroform to kill bacteria and spun at 800 x g for 5 minutes. Phage
was collected from supernantant. Free virus was then assayed by plaque assays in 24-well tissue
culture plates.
MOI study:
Cells in exponential growth were infected with five dilutions of viral lysate stock (0, -1, 2, -3, and -4 log dilutions). Phage A stock titer was approximately 107 PFU/mL, and phage C
stock was approximately 109 PFU/mL. Infected cells were diluted 1:100 into fresh liquid media
before being incubated incubated at 37°C. For the A-phage study, OD600 measurements were
taken during the course of the infection. Cultures were collected upon viral lysis and treated
with chloroform to extract intracellular and adsorbed phage particles. Samples for microscopy
were purified of cell debris by centrifugation and filtration through 0.2 um membrane filters
before being fixed with 2% paraformaldehyde. Samples for infectivity (plaque) assays were only
centrifuged.
Morphology by transmission electron microscopy (TEM):
Phage stocks and infected cells were fixed with 4% paraformaldehyde and placed onto
formovar coated nickel grids. Samples were stained with 4% uranyl acetate solution for 30
seconds. Grids were visualized on a trasmission electron microscope (Zeiss) at varying
resolutions and captured using a digital camera.
Lysogenic inductions:
Host cultures A and C and six other bacterial isolates were tested for the presence of
lysogenic phage by adding 0.1 mg/uL mitomycin C. Cleared cultures were collected, filtered,
and fixed for TEM visualization.
Results and Discussion:
Host phylogeny:
Both A and C host had 16S sequences 99% similar to Pseudoalteromonas species (>99%
identical to each other and databased Pseudoalteromonas species).
DNA extraction and sequencing:
CsCl gradient extraction was performed twice, yielding two tubes of DNA for the first try
(CsCl 1-1 an 1-2) and one for the second (CsCl 2-1). Extractions without CsCl were also
performed yielding two tubes each for phenol-chloroform (PC 1 and 2) and CTAB-phenolchloroform (CTAB 1 and 2). All DNA aliquots were positive for bacterial 16S DNA (Figure 1),
which was revealed by sequence to be the Pseudoalteromonas host. Four DNA aliquots with the
lowest level of 16S bacterial DNA as quantified by gel-electrophoresis (CsCl 1-1, CsCl 1-2, PC
2, and CsCl 2-1) were digested with a 10-fold amount of restriction enzyme BanI (sequence
G^GYRCC) (New England Biolabs) and purified with phenol-chloroform extraction and sodium
acetate-ethanol precipitation. Purified DNA was visualized on a gel (Figure 2).
Figure 1: Gel of 16S PCR of extracted DNA. The first set of three is 1:10, 1:100, and
1:1000 dilutions of CsCl 1-1. The second set of three includes the same dilutions for CsCl
1-2. The last two lanes have 1 uL of the original DNA extractions
Figure 2: Gel of purified restricted DNA from phage extraction. Lane 1 is CsCl 1-1. Lane
2 is CsCl 1-2. Lane 3 is Phenol-chloroform extraction PC2. Lane 4 is CsCl 2-1
My original intent was to generate blunt ends and clone into puC19 vector, but due to the
lack of time and DNA ligase, the restricted DNA was instead adenilated and clones into the
TOPO-kit vector (Invitrogen). The resulting clone library yielded 38 sequences with lengths
between 413 and 806 nucleotides in length. None of the sequences formed coherent contigs with
one another when run through CLC Workbench Assembler.
22 high-quality sequences with vector sequences removed were compared to NCBI’s ‘nt’
database using BLASTN, and none of the sequences had BLAST hits longer than 36 nucleotides.
The sequences were also compared to NCBI’s nr protein database using BLASTX. Of the 16
sequences with BLASTX hits, 4 were similar to phage genes, 7 were similar to
Pseudoaltermonas genes, and 5 were similar to other bacterial genes (Table 1). The presence of
sequences related to host genes could be caused by contamination with host DNA in the DNA
extraction or by legitimate incorporation and evolution of host genes in the phage genome. 6 of
the high-quality sequences submitted to BLASTX had no similarity to any proteins in the
database. This fits with the trend that many environmental phage sequences are novel1. Figure 3
and Table 1 summarize BLASTX hits and sequence identity.
Figure 3: Sequence identity and coverage for BLASTX hits from different sourc
Table 1: BLASTX protein top hits for 16 sequences
identity
60
57
85
83
47
86
16
63
55
40
56
57
53
69
36
40
coverage
83
90
85
92
58
93
79
72
44
72
44
89
90
96
88
84
protein hit
GGDEF domain (Alteromonas)
hypothetical protein (Pseudoalteromonas
glycine cleavage aminomethyltransferase (Alteromonas)
valine tRNA synthase (Pseudoalteromonas)
uncharacterized orphan protein (Pseudoalteromonas)
Double-use DNA protein (Pseudoalteromonas)
hypothecial protein (Pseudoalteromomas)
phage tail protein
Vibrio phage protein
Salmonella phage hypothetical protein
Escherichia phage tail protein
ABC transporter
ribosomal Shewanella protein
lyase/N-acetylglutamate synthase
hypothetical Sulfitobacter protein
hypothetical Sulfitobacter protein (a different one)
source
Host
Host
Host
Host
Host
Host
Host
Phage
Phage
Phage
Phage
Other
Other
Other
Other
Other
Cross-infectivity:
In light of the close phylogenetic relationship of the two host strains, both strains were
infected with both viruses to assess whether isolated phage can infect the opposite strain. Aphage was found to infect both isolate A and isolate C and resulted in significant clearing (Figure
4). The turbidity cultures infected with C-phage did not differ from control cultures. This is
particularly curious because C-phage was previously found to cause lytic infection in isolate C.
This discrepancy may be caused by different environmental parameters inducing C-phage to
enter the lysogenic cycle. It would be very interesting to conduct further studies to determine if
the lysogenic/lytic decision can be predicted under specific environmental conditions, substrate
limitation, and MOIs.
Figure 4: Optical density after 24 hours of isolates A and C infected with both phage
Adsorption kinetics:
The adsorption curve for the C-phage was obtained for the first 10 minutes after infection
(Figure 5). The y-axis is the number of phage that remain in solution (not adsorbed to cells) after
a given time. The reason for the low PFU count at 1 minute is unknown, and titers for the 7
minute sample could not be obtained.
If the first point is discarded, and the remaining data plotted on log-scale, a linear
regression line can be used to find the adsorption rate (Figure 6). Phage adsorption typically
follows the first-order kinetics according to P = Poe-kNt, where P is the titer of free phage at time
t, Po is the original phage titer, k is the adsorption constant in mL/min, N is the bacterial
concentration at the time of infection, and t is the time after infection. The kinetics equation can
be rearranged to yield (ln Po – ln P)/t = kN. The left side of the last equation is the slope of the
graph ln(P) versus time. Thus k is calculated as 2.70 x 10-9 mL/min (assuming a bacterial
density of 108 cells/mL at 1.0 OD600). This constant accounts for both diffusion and attachment
rates and is specific to the phage, host, and adsorption conditions17. This one value does not
offer much insight into the adsorption process, but it would be interesting to characterize
adsorption constants at different MOIs, environmental conditions, and substrates to learn more
about adsorption mechanisms in the phage.
Figure 5: Infective particles remaining un-adsorbed versus time after infection
Figure 6: Linear fitting of first-order adsorption kinetics for 10 minutes of C-phage
infection in isolate C
One-step growth curve:
A one-step growth curve was attempted three times with samples taken every 10 minutes
for 100 minutes, but the plaque assays yielded no infection. Failure to maintain detail notes
regarding PFU assay set-up disables intelligent diagnosis of the problem. It is possible that some
environmental condition induced lysogeny in the C-phage and thus led to an absence of plaques.
MOI study:
Optical density of infected cultures increased with bacterial growth over the first 10 hours
of infection (Figure 7). Subsequently, density dropped due to cell lysis in the order of highest to
lowest MOI. 24 hours after the incubation began, the optical density of all cultures had dropped
by a factor of 2 from maximum.
Two control tubes that were not inoculated with virus was incubated simultaneously. OD
measurements from these tubes were alternately taken at the same time points. The measured
OD for the control tubes also appeared to drop at a similar rate as the 10-4 dilution. This is a
remnant of one control culture remaining turbid and one culture clearing, probably as a result of
phage contamination. In future analyses I will be sure to use several control cultures and to
measure them all to avoid this problem.
Figure 7: Optical density at 600nm recorded over the course of MOI experiment for Aphage.
Figure 8: PFU concentration of viral lysates collected from cells infected at different
dilutions of viral stock
The number of infective phage particles in the lysate increased with decreasing MOI for
both phages A and C (Figure 8). This trend could be a result of several different things.
Additionally, at MOIs greater than 1 (dilution 0 for A-phage and dilution -2 for C-phage), phages
can inhibit the attachment of other phages, so that increasing MOI would not be expected to
yield a higher titer. The cultures with low MOI took longer for cells to lyse, so more generations
of phage could be produced and a higher final titer could be obtained.
SYBR green staining of all MOI samples resulted in viral density to high to count even
after 109 dilutions.
Lysogenic induction:
Of the eight cultures tested for lysogenic induction, two (D and E) exhibited clearing
after incubation with mitomycin C. Lysates were filtered and used to re-infect host cultures in
the absence of mitomycin C, and no clearing was observed. The lysogenic phage for E was
imaged on TEM (Figure 9). The phage appeared hexagonal on a plane and were about 40 nm
across. Some of the phage had a long tail about 90 nm long. Phylogenic information for isolate
E was not obtained due to lack of time.
Figure 9: Transmission electron microscopy images of tailed lysogenic phage from isolate
E. Scale bar is 100 nm on left and 20 nm on right.
TEM of A and C cells and phages:
TEM of A and C phages revealed both to have a isocahedral capsid. No tails were seen
on A-phage, and very long filamentous tails may have been seen on C-phage (Figure 10). A
sample of isolate A host cells infected with virus at 15 and 35 minutes was also stained and
visualized (Figure 11). Many detached flagella were seen, and they were probably disconnected
during culture manipulation and staining. Phage were commonly seen attached to flagella
(Figure 11.B). Several host cells appeared to be undergoing lysis, but resolution was not
sufficient to visualize phage infection (Figure 11.C).
Figure 10: TEM visualization of C-phage
A
B
Figure 11: TEM of isolate A cells
infected with A-phage for 15 minutes
before fixation. A.) A cell B.)
phage-like particles adsorbed to
detached flagella C.) A cell
undergoing lysis D.) Flagellated A
cell with phage particles nearby
C
D
Conclusions:
Phage are fickle. I successfully characterized adsorption of phage to cells under one
environmental condition, but more information could be gained from finding adsorption
constants at differing conditions. I determined that infection at lower multiplicity of infection
generally results in more total infective virus produced, but was unable to characterize the
relationship between MOI and the ratio of infective to non-infective particles due to a lack of
accurate SYBR green epifluorescence counts. I also failed at obtaining results from a one-step
growth curve because the resulting phage lysate did not cause lysis in plaque assays. This may
indicate that the C-phage can be lysogenic, or may be a result of poor documentation of plaque
assay set-up on my part. The pursuit of a more efficient plaque assay technique using 96-well
plate dilutions and 24-well plaque assays was successful, and allowed infective phage counts of
many samples at many different dilutions without the onset of disabling hand injury due to too
much pipetting. TEM imaging was generally successful, but it would have been beneficial to
spend more trying different staining conditions and scope voltage. In general, I learned a great
deal about phage culture and DNA manipulation and hope to be able to use this knowledge when
I return to my CU-Boulder to begin my Ph.D. thesis project.
Regrets/Suggestions for future phage projects:
If I were given the opportunity to start the project again with the knowledge gained along
the way (or if someone in future years were to read this report looking for ideas/suggestions for a
phage project), I would make several changes.
•
•
•
•
Host characterization: I would spend some time obtaining more information about
the host organism.
o Ratios of viable cells to optical density would be useful in setting up
plaque assays
o Respiration rates and growth curves would also be useful
DNA extraction and sequencing:
o Try MoBIO’s lambda phage extraction kit (I didn’t know it existed at the
time of this report) to see if same results are obtained and if bacterial
contamination still occurs.
o Try sequencing blunt ends (digest or sonicate or nebulize and treat with
blunting kit) and cloning into puC19 or other vector.
o Try cutting specific lengths of DNA fragments from gel and only
sequencing fragments between 400 and 1000 nt long.
o Try different restriction enzymes to obtain different DNA fragments.
Especially if you want to select for lengths.
Plaque assays: The use of 24-well plates for plaque assays was very successful.
However, plaque assays ceased to work for the last 3 days of the project for
unknown reasons. I would take more detailed notes about what OD, phage
dilution, and volumes are used in plaque assays to determine what works and
what does not.
SYBR counts:
o Don’t be put off by diluting things by 1010 or more. Phage are very
numerous in lysate stocks!
•
•
o Try more filters to get a better feel for what produces consistent results
and how much phage is appropriate.
Adsorption kinetics: perform experiment and calculate adsorption constants for a
range of environmental parameters
Lysogeny: set up differing substrate limitation situations under different MOI and
environmental conditions to see when lysogeny occurs.
o Are there some conditions that select for lytic vs. lysogenic phage?
o Is the C-phage lysogenic, and if so under what conditions?
Acknowledgements:
Thanks to the Moore Foundation and the Selman A. Waksman endowed scholarship for
financial support. I also would like to thank my home Ph.D. advisors, Mark Hernandez and
Norm Pace for both financial and academic support. I also owe gratitude to Bekah Ward for
TEM help, Gil Sharon for phage culture insights and moral support during DNA extraction,
Annie Rowe for PFU assay help, Chuck Pepe-Ranney and Libusha Kelly for bioinformatics,
Heather Fullerton and Gargi Kulkarni for cloning advice, Hilary Morrison and the Sogin lab for
molecular materials. Thanks to MBL course directors and to all my fellow students who have
shared invaluable insights and memories with me this summer. What what.
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Appendix A: Phage DNA extraction protocol
DNA extraction of phage lysate (for 200 mL lysate):
1.)
Add 1M NaCl and 10% polyethylene glycol was added to viral lysate and
incubate on ice for one hour. Pellet phage particles by centrifuging for 30
minutes at 11,000 g.
2.)
Discard supernatant and let pellet dry diagonally inverted in hood for 5
minutes. Re-suspend in SM buffer (1.6 mL/100 mL) and incubate to let pellet
dissolve for 1 hour.
3.)
Add 1 volume chloroform, vortex 30 seconds, and centrifuge for 15 minutes
at 3,000g. Transfer aqueous phase to a clean tube.
4.)
Layer 1 mL of the heaviest CsCl layer first (1.7 mg/mL = 1.4013 refractivity),
and then proceed with the other two layers (1.5 mg/mL = 1.3833 refractivity
and 1.35 mg/mL = 1.3701 refractivity). Add phage solution (sup) to top of
tube, being careful to not disturb the layers. Top off tube with SM buffer.
Weigh to ensure that tube weights are within 1 mg.
5.)
Centrifuge 2 hrs at 60,000g at 4C. (TLA110 rotor in Beckman UltraMAX at
33,200 rpm).
6.)
Set up the tube in a stable rack and use a syringe to take the phage layer
(orange or white) from the tube (about 0.5 mL per tube)
7.)
Treat with 2.5 U of DNAase I per mL sample (1 uL of 2500 U/mL per mL
sample). Incubate 1 hour at 37C.
8.)
Add 0.1 volumes 2M/0.2M Tris/EDTA at pH 8.5, 0.01 volumes 0.5M EDTA
at pH 8.0, 1 volume formamide. Incubate at room temperature for 30 minutes
9.)
Precipitate by adding 2 volumes 100% ethanol. Pellet DNA for 20 minutes at
10,000g at 4C.
10.)
Wash pellet twice with 70% ethanol, spinning for 5 minutes in between.
11.)
Resuspend in 800 uL of 0.05M Tris-CL at pH 8 (STOP POINT)
12.)
Add 1 volume phenol and vortex for 20 minutes to break up phage. Spin 10
minutes at 20,000g and save top layer.
13.)
Repeat phenol extraction
14.)
Add 1 volume chloroform and vortex for 20 minutes to break up phage. Spin
10 minutes at 20,000g and save top layer.
15.)
Repeat chloroform extraction
16.)
Precipitate DNA by adding 20 uL (0.1 volume) of 3M sodium acetate at pH
4.8 and 2 volumes 100% ethanol. Spin 10 minutes at 16,000g.
17.)
Remove supernantant and wash pellet with 70% ethanol. Air-dry.
18.)
Resuspend pellet in 0.05M Tris-Cl buffer (pH 8.0).