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How does one generate single-stranded DNA (ssDNA) from the pGEM®-T Vector?
The pGEM®-T (Cat.# A3600, A3610) and
pGEM®-T Easy (Cat.# A1360, A1380) Vectors* are phagemids that contain an f1 ori
region (derived from the filamentous bacteriophage f1 origin of replication), which allows
for the production of single-stranded phagemid DNA.
To generate ssDNA from a pGEM® Vector, the phagemid must be propagated in
an E. coli strain containing an F´ episome. The F´ episome encodes the proteins
required for the sex pili on the surface of the E. coli that serve as attachment
and entry sites for the filamentous bacteriophages, M13, f1 and fd. Following infection of
such a strain with a helper filamentous bacteriophage (e.g., R408; Cat.# P2291), the
plasmid then enters the f1 replication mode and the resulting ssDNA is exported from the
cell as an encapsulated virus-like particle. The ssDNA is purified from the supernatant by
simple extraction and precipitation.
Helper phage
are modified phage that provide components required for propagation, packaging and
secretion of f1 ori-containing plasmids as ssDNA in viral particles. Helper
Phage R408 (Cat.#
P2291) allows the preferential secretion of single-stranded plasmid DNA over the phage
ssDNA, thereby increasing the absolute yield of plasmid ssDNA. |
Brief protocol for the isolation of ssDNA from pGEM®
Vectors
- Prepare an overnight culture of cells containing pGEM® Vector (or
recombinant vector) by picking individual ampicillin-resistant colonies from a fresh
plate. Inoculate 1-2ml of LB broth containing 100µg/ml ampicillin and shake at
37°C.
- The next morning, inoculate 50ml of LB broth containing 100µg/ml ampicillin with
1ml of the overnight culture. Shake vigorously with good aeration at 37°C for 1 hour in a
250ml flask.
- Infect with helper phage R408 at an m.o.i. (multiplicity
of infection) of 10-20; i.e., add 10-20 helper phage particles per cell. Continue shaking
for 6 hours with vigorous agitation. Good aeration of the culture is important for
obtaining high yields of ssDNA.
- Harvest the supernatant by pelleting the cells at 12,000 x g for 15
minutes. Pour the supernatant into a fresh tube and centrifuge again for 15 minutes (at
4°C).
Note: Pelleting the cells twice is important for reducing the level of
contaminating cellular nucleic acid in the supernatant. An additional level of purity of
ssDNA can be attained by treating the supernatant with both DNase I and RNase A (10µg/ml
each) for 15 minutes at 37°C.
- Precipitate the phage by adding 0.25 volume of phage precipitation solution
(3.75M ammonium acetate [pH7.5] and 20% polyethylene glycol 8000) to the supernatant.
Leave on ice for at least 1 hour (or overnight) and centrifuge for 15 minutes at 12,000 x g.
Thoroughly drain the supernatant.
- Resuspend the pellet in 400µl of TE buffer (pH 8.0).
- Add 400µl of phenol:chloroform:isoamyl alcohol (25:24:1) saturated with TE
buffer. Vortex for 1 full minute and centrifuge for 5 minutes at 12,000 x g.
- Transfer the upper, aqueous phase to a fresh tube and repeat the phenol
extraction as in Step 7. It may be necessary to repeat the extractions several times until
there is no visible material at the interface.
- Transfer the upper, aqueous phase to a fresh tube and add 400µl of
chloroform:isoamyl alcohol (24:1). Vortex for 1 full minute and centrifuge as in Step 7.
Repeat this procedure once more.
- Transfer the upper, aqueous phase to a fresh tube and add 200µl of 7.5M ammonium
acetate and 1.2ml of 100% ethanol. Mix and leave at -20°C for 30 minutes to precipitate
the phagemid DNA.
- Centrifuge at 12,000 x g for 15 minutes, remove the supernatant and
carefully rinse the pellet with ice-cold 70% ethanol. If the pellet is disturbed,
centrifuge again (for 2 minutes). Drain the tube and dry the pellet under vacuum.
- Resuspend the DNA in 20µl of water.
Multiplicity of infection or
m.o.i.: How to determine the volume of phage to add to obtain the correct
m.o.i.
The volume of phage to add to arrive at an m.o.i. of 10-20 can be
calculated by assuming that the cell concentrations of the starting cultures range from
about 5 x 107 to 1 x 108 cells/ml (OD660 = 0.1-0.3). To
infect at an m.o.i. of 10-20 requires 5 x 108 to 2 x 109 phage/ml or
10-40µl of phage/ml of cells using a 5 x 1010pfu/ml stock of helper phage. An
m.o.i. as high as 100 will not alter the yield of ssDNA. |
*Products may be covered by pending or issued
patents. Please visit our patent and trademark web page for more
information.
pGEM is a trademark of Promega Corporation and is registered with the U.S. Patent and
Trademark Office.
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