pRc/RSV - Invitrogen

pRc/RSV
Version C
171023
28-0051
U.S. Headquarters: European Headquarters:
Invitrogen Corporation Invitrogen BV
1600 Faraday Avenue De Schelp 12, 9351 NV Leek
Carlsbad, CA 92008 The Netherlands
Tel: (800) 955-6288 Tel: +31 (0) 594 515 175
Fax: (760) 603-7201 Fax: +31 (0) 594 515 312
E-mail: tech_service@invitrogen.com E-mail: tech_service@invitrogen.nl

pRc/RSV
Supercoiled Vector
Catalog No. V780-20
Contents
pRc/RSV, 20mg of supercoiled vector, lyophilized in 10mM Tris-HCl, pH7.4, 1mM EDTA.
TOP10F' bacterial strain, supplied as a stab.
Reconstitute the vector in sterile water to a final concentration of 1mg/ml.
Store the reconstituted vector at -20°C.
Stabs should be stored at +4°C.
Description
pRc/RSV is a 5.2kb vector designed for high-level stable expression in eukaryotic hosts. The RSV
promoter has shown high-level expression in a wide range of mammalian cells. This vector will replicate
episomally in cell lines that are latently infected with SV40 or which express the SV40 large T antigen (e.g.
COS7). High-level stable and non-replicative transient expression can be carried out in most mammalian
cells.
Features
Features-for eukaryotic expression
• RSV LTR (Rous sarcoma virus long terminal repeat) promoter and enhancer which will express inserted
genes from the first ATG codon (provided by insert).
• Multiple cloning site polylinker
• Bovine growth hormone (BGH) polyadenylation signal for polyadenylation of transcribed mRNAs
• SV40 origin for transient episomal replication in cells expressing SV40 large T antigen
• Neomycin resistance marker, expressed from the SV40 early promoter for the selection of stable
transformants in the presence of G418
Features for growth and maintenance in E.coli:
• b-lactamase gene to confer ampicillin resistance
• ColE1 origin of replication from pUC19 for high copy number growth
• f1 origin for rescue of single-stranded DNA (for mutagenesis or single-strand sequencing)
• Inverted BstX I sites for efficient cDNA library construction using Invitrogen's non-palindromic cloning
strategy
Note: There is an ATG upstream from the Xba I and Apa I sites within the polylinker.
E. coli Strains
TOP10F' is provided for growth and maintainence of this plasmid. TOP10F' is a recombination negative strain
designed for stable replication of high copy number plasmids. Since TOP10F' carries a F' episome, it is suitable for
single strand phagemid rescue. This strain is provided as a convenience for those who do not have access to
other E.`coli strains. Many E.coli strains are suitable for the growth of this vector including DH5aF', JM109, and
INVaF'. In general, grow vectors containing inserts in E. coli strains which are recombination deficient (recA-).
Instructions for growth of TOP10F' stab:
Using a flame sterilized loop, streak from the TOP10F' stab onto LB agar plates supplemented with
15mg/ml of tetracycline. Incubate the plates at 37°C overnight.
On the following day, pick well-isolated, single colonies from these plates for the preparation of frozen
stocks and competent cells.
Any standard protocol for competent cell preparation and transformation is suitable for use with this strain.
Genotype of TOP10F'
F'{lacIqTn10(TetR )} mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15 DlacX74 deoR recA1 araD139 D(ara-leu)7697
galU galK rpsL endA1 nupG
Selected Genotype Explanations
• recA1 is for increased stability of inserts, reducing recombination by 1000 fold.
26-0026 C
US Headquarters: European Headquarters:
800-955-6288 2 +31 (0) 594 515 175

pRc/RSV
Supercoiled Vector
Catalog No. V780-20
• endA1 is for improved quality of minipreps.
• hsdRMS eliminates cleavage of recombinant plasmid by the endogenous EcoR restriction system.
• deoR: A mutation that has been found to improve plasmid transformations, particularly of large constructs (up
to 66kb).
• Carries the F' episome which is required for pili formation. F' is required for infection by helper phage (e.g.
R408, M13K07) for rescue of single strand DNA from vectors containing f1 (M13) origins. Note that this F'
carries the tetracycline resistance marker. For maintenance of the F', these cells must be plated on media
containing tetracycline (15mg/ml).
• The lac I q encodes lac repressor which enables repression of genes containing the lacO in the promoter
region. In the presence of lac I q encoded repressor, such lacO containing genes remain silent until induction
(de-repression)with IPTG.
• LacZDM15 encodes a b-galactosidase (W-fragment) which has a N-terminal deletion of 146aa. The LacZDM15
gene product is not functional alone but can be complemented by "a-complementation" by the a part (first
146aa, encoded by Lac Za). The a part of b-galactosidase is usually encoded on a plasmid or phage (in trans)
downstream of the IPTG inducible Lac promoter. a-complementation is the basis for blue-white screening. A
functional b-galactosidase catalyzes the white –> blue reaction of the chromogenic substrate, X-gal, resulting in
white "positive" and blue "background" colonies. Electrocompetent and chemically competent Top10F' cells
are available from Invitrogen as follows:
Catalog No. Description
Efficiency Aliquots
C665-55 Electrocompetent Top10F' 1 x 109 5 x 80ml
C665-11 Electrocompetent Top10F' 1 x 109 10 x 80ml
C665-03 Ultracomp chemically competent TOP10F' 1 x 108 5 x 300ml
L1165-01 Library Size Transformation Kit 3 x 108 5 x 300ml
Chemically competent TOP10F' (5 x 1ml)
L1165-55 Library Size Electroporation Kit - TOP10F' 1 x 109 10 x 80ml
(also includes chemically competent cells for test ligations) (5 x 300ml)
Recommendations
A protocol for the purification of single-stranded DNA has been provided. Several other protocols for this
procedure may be found in references such as "Molecular Cloning" by Maniatis or "Current Protocols in
Molecular Biology". There are two strains of helper phages recommended for this procedure, R408 (Cat#
R568-01) and M13K07 (Cat# R137-01).
Transfection of Mammalian Cells
Many different methods for transfection of mammalian cells exist. Some of those are Calcium Phosphate,
DEAE-Dextran or Liposome mediated, and electroporation. For stable transfections, the Calcium
Phosphate method is the most widely used.
Invitrogen's Calcium Phosphate Transfection Kit is a simple and reliable method for transfection of almost
any adherent cell type. Invitrogen also offers an electroporator which is ideal for mammalian
electroporation of non-adherent cells, when used with a compatible power supply (not included). Please
call for additional information on these products.
Catalog No.
Description
K2780-01 Calcium Phosphate Transfection Kit 75 reactions
S1650-02 ElectroPorator 1unit
Calcium Phosphate Transfection (Wigler, M., Silverstein, S., Lee, L-S, Pellicer, A., Cheng, Y-C, and Axel,
R. 1977).
Electroporation (Chang, D.C, 1989), (Chu, G., Hayadawa, H., and Berg, P. 1987) and (Guide to
Electroporation and Electrofusion 1992).
26-0026 C
US Headquarters: European Headquarters:
800-955-6288 3 +31 (0) 594 515 175

pRc/RSV
Supercoiled Vector
Catalog No. V780-20
Neomycin (G418) Selection in Mammalian Cells
Basis for selection:
G418 blocks protein synthesis in mammalian cells by interfering with ribosomal function. It is an
aminoglycoside, similar in structure to neomycin, gentamycin, and kanamycin.
The bacterial aminoglycoside phosphotransferase (APH) gene is derived from Tn5. Expression in
mammalian cells results in detoxification of G418.
Selection conditions:
G418 is available from Gibco/BRL. Use 100 to 800mg/ml of G418 in complete medium. G418 should be
prepared in a highly buffered solution (e.g. 100mM HEPES, pH 7.3) so that the addition of the drug does
not alter the pH of the medium.
Varying concentrations of G418 should be tested as cells differ in their susceptibility to killing by G418.
Different lots of G418 can have different potencies, causing many investigators to buy a large amount of
one lot to standardize selection conditions. G418 concentration should be calculated using the amount
of active drug (usually indicated on the label) so that variance is controlled.
Cells will divide once or twice in the presence of lethal doses of G418, so the effects of the drug take
several days to become apparent. Complete selection can take up to 3 weeks of growth in selective
media.
Protocols
Preparation of Single-stranded DNA from Invitrogen's Phagemids
Infection with Helper Phage
There are two strains of helper phage recommended, R408 (Cat# R568-01) and M13K07 (Cat # R137-
01). When using M13K07 make sure that the helper phage was selected with Kanamycin to ensure
retention of the proper phenotype.
1. Inoculate 3.0ml of SOB media with 0.2ml of an overnight culture that is male-specific (contains the F'
episome coding for pili production) and carries a plasmid that has the f1 or M13 ori allowing singlestranded
rescue.
2. Allow the inoculant to reach OD600 = 0.3 (approximately 40 minutes at 37°C) and infect with helper
phage at an MOI (multiplicity of infection) of 20:1 (phage:cells).
Note: When the OD600 = 0.3, there will be approximately 2.5 x 108 cells/ml.
3. Grow for 6 to 8 hours at 37°C with vigorous shaking (at temperatures less than 34°C, the F-pilus is not
produced and therefore does not allow helper phage infection).
4. Pellet the cells at 2000 rpm for 10 minutes. If you need to store the rescued material, heat the
supernatant to 60-70°C for 30 minutes to inactivate the bacterial cells (do not add chloroform). Phage
are stable for long periods of time at 4°C.
Purification of Single-stranded DNA
There are several protocols for the purification of single-stranded DNA. The following is only one of
several and produces high yields of single-stranded DNA for sequencing or mutagenesis.
1. Transfer the supernatant to a fresh microcentrifuge tube. Treat the supernatant with 1mg/ml of
RNase and 10mg/ml of DNase and then incubate at 37°C for 10 minutes.
2. Add 1/4 volume of a solution of 3.5M ammonium acetate and 20% polyethylene glycol (MW 6000).
3. Incubate the solution on ice for at least 10 minutes. Microcentrifuge the tube for 5 minutes.
26-0026 C
US Headquarters: European Headquarters:
800-955-6288 4 +31 (0) 594 515 175

pRc/RSV
Supercoiled Vector
Catalog No. V780-20
4. Pour off the supernatant (there should be a small pellet) and wipe the inside of the tube, being
careful not to disturb the pellet.
5. Resuspend the pellet in 200ml of TE buffer (10mM tris, pH 8.0, 1mM EDTA) and vortex the tube
vigorously to complete resuspension.
6. Add 200ml of buffer saturated phenol (neutralized with 0.1M Tris, pH 8.0), vortex the tube vigorously
for 30 seconds and spin in a microcentrifuge for 5 minutes.
7. Using a pulled pasteur pipette, carefully remove the bottom organic layer and discard. Save the
aqueous layer and the interphase in the original tube. Much of the DNA can be bound to proteins
at the interphase. Repeat this phenol extraction one more time.
8. Extract one time with 200ml phenol-chloroform (1:1) using the same technique as in Steps 6 and 7.
Save the aqueous and the interphase in the original tube.
9. Extract twice with 200ml of chloroform as before. On the final extraction, transfer the aqueous (top)
layer to a fresh microcentrifuge tube leaving the interphase behind.
10. Add 150ml of 7.5M ammonium acetate and 1ml of 95% ethanol. Mix the solution thoroughly.
11. Incubate the solution at -70°C for 30 minutes then spin in a microcentrifuge for 10 minutes at room
temperature.
12. Pour off the supernatant and add 1ml of cold (-20°C) 95% ethanol. Spin the tube in a microcentrifuge
for 5 minutes.
13. Pour off the supernatant and vacuum the tube dry. There may not be a visible pellet but rather a haze
on the side of the tube, this is the DNA.
14. Resuspend the DNA in 10-20ml of 10mM Tris HCl, pH 7.5, and store at -20°C.
15. Run 1-5ml of the DNA on a 1% agarose gel. Both the phage and the phagemid DNA should be
visible and roughly at a 1:1 ratio. To calculate the amount of single-stranded DNA, determine the
OD260, calculate the amount of total DNA and estimate the percentage that is single-stranded from
the agarose gel.
16. A faint band may appear below the major DNA bands, indicating single-stranded DNA that is nicked
and therefore linear. This band will get more intense upon storage and subsequent freezing and
thawing of the DNA. It is difficult to detemine where the helper phage will run on a 1% agarose gel,
but it is typically between 4 and 6kb. The yield of single-stranded phagemid DNA is less than with
clones in an M13 vector. Adjustments should be made for amounts required for sequencing.
References
Calcium Phosphate Transfection:
Wigler, M., Silverstein, S., Lee, L-S, Pellicer, A., Cheng, Y-C, and Axel, R. (1977) "Transfer of Purified
Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells." Cell 11: 223-232.
Neomycin Selection:
Southern, P.J. and Berg, P (1982) "Transformation of mammalian cells to antibiotic resistance with a
bacterial gene under control of the SV40 early region promoter." J. Mol. Appl. Gen. 1:327-341.
Current Protocols in Molecular Biology (1990) ed. Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore,
J.G. Seidman, J.A. Smith, K.Struhl. pub. Greene Publishing Associates and Wiley-Interscience.
This is a large two volume set)
Molecular Cloning, A Laboratory Manual, 2nd Edition. (1989) Sambrook, Futsch, Maniatis. Cold Spring
Harbor Laboratory Press. This is a three volume set.
26-0026 C
US Headquarters: European Headquarters:
800-955-6288 5 +31 (0) 594 515 175

pRc/RSV
Supercoiled Vector
Catalog No. V780-20
RSV LTR Promoter References:
Gorman, C.M., Merlino, G.T., Willingham, M.C., Pastan, I., Howard, B.H., (1982) The Rous Sarcoma Virus
Long Terminal Repeat is a Strong Promoter when Introduced into a Variety of Eukaryotic Cells by
DNA-mediated Transfection. PNAS 79: 6777-6781.
Yamamoto, T., De Combrugghe, B., and Pastan, I., (1980) Identification of a Functional Promoter in the
Long Terminal Repeat of Rous Sarcoma Virus. Cell 22: 787-797.
Norton, P.A., and Coffin, J.M., (1987) Characterization of Rous Sarcoma Virus Sequences Essential for
Viral Gene Expression. J. Virol. 61: 1171-1179.
pRc/RSV Citations:
Wen-chang Lin and Lloyd A. Culp (1991) Selectable Plasmid Vectors with Alternative and Ultrasensitive
Histochemical Marker Genes BioTechniques 11: 344-351.
Jean-Christophe Renauld, Catherine Druez, Abdenaim Kermouni, Frederic Houssiau, Catherine
Uyttenhove, Emiel Van Roost, and Jacques Van Snick (1992) Expression Cloning of the Murine and
Human Interleukin 9 Receptor cDNA PNAS 89: 5690-5694.
James T. Campanelli, Werner Hoch, Fabio Rupp, Thane Kreiner, and Richard H. Scheller (1991) Agrin
Mediates Cell Contact-Induced Acetylcholine Receptor Clustering Cell 67: 909-916.
26-0026 C
US Headquarters: European Headquarters:
800-955-6288 6 +31 (0) 594 515 175