Catalog #216201 Revision A
For In Vitro Use Only
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Materials Provided???? 1 Storage Conditions ???? 1 Vector Features ???? 2 Applications???? 2 The pSG5 Vector???? 3 Preparation of Host Strain ???? 4 Preparation of a ?C80?ãC Glycerol Stock ???? 4 Preparation of Media and Reagents ???? 4 MSDS Information???? 5
pSG5 Vector MATERIALS PROVIDED
Material Provided pSG5 vector AG1 strain*, glycerol stock Quantity 20 ?Ìg 1 tube
* recA1, endA1, gyrA96, thi-1, hsdR17, (rk?C, mk+), supE44, relA1, (uncharacterized mutation improves transformation efficiency)
pSG5 vector: ?C20?ãC AG1 bacterial glycerol stock: ?C80?ãC
Agilent Technologies, Inc. 2008
The pSG5 Vector is a eukaryotic expression vector constructed by combining pKCR2 and the Stratagene pBS vector. Because of the high copy number of this plasmid, large quantities of double-stranded DNA are obtained.
The pSG5 is useful for both in vitro and in vivo expression. Expression in vivo is achieved via transient expression in a variety of cell lines (highest level of expression is obtained following transfection of a cell line expressing the T antigen). The f1 origin allows rescue of ssDNA for use in mutagenesis and sequencing. The SV40 early promoter and polyadenylation signal promotes expression in vivo, and the T7 bacteriophage promoter facilitates in vitro transcription of cloned inserts. The ?Â-globin intron II allows splicing of expressed transcripts. To ligate the gene of interest into the pSG5 vector, use the unique restriction sites EcoR I, BamH I, and Bgl II (downstream from the promoter).
The pSG5 Vector
f1 ori P SV40/SV40 ori
EcoR I BamH I Bgl II
SV40 pA ampicillin pUC ori
Feature SV40 promoter and SV40 origin of replication ?Â-globin intron T7 promoter EcoR I BamH I Bgl II SV40 polyA signal pUC origin of replication ampicillin resistance (bla) ORF f1 origin of ss-DNA replication
Nucleotide Position 28?C366 395?C967 1022?C1040 1043 1049 1055 1069?C1202 1342?C2009 2160?C3017 3587?C3893
Figure 1 Circular map and features of the pSG5 vector. The complete
sequence and list of restriction sites is available at
PREPARATION OF HOST STRAIN
The host strain has been sent as a glycerol stock. For the appropriate media and plates, please refer to the following table:
Bacterial strain AG-1 Plates for bacterial streak LB Media for glycerol stock LB
On arrival, prepare the following from the glycerol stock: Note Do not allow the contents of the vial to thaw. The vials can be stored at ?C20 or ?C80?ãC, but most strains remain viable longer if stored at ?C80?ãC.
Revive the stored cells by scraping off splinters of solid ice with a sterile wire loop. Streak the splinters onto an LB plate.
Restreak the cells fresh each week.
Preparation of a ?C80?ãC Glycerol Stock
1. In a sterile 50-ml conical tube, inoculate 10 ml of the appropriate liquid media with one or two colonies from the plate. Grow the cells to late log phase. Add 4.5 ml of a sterile glycerol?Cliquid media solution (5 ml of glycerol + 5 ml of liquid media) to the bacterial culture from step 1. Mix well. Aliquot into sterile centrifuge tubes (1 ml/ tube).
This preparation may be stored at ?C20?ãC for 1?C2 years or at ?C80?ãC for more than 2 years.
PREPARATION OF MEDIA AND REAGENTS
LB Broth (per Liter)
10 g of NaCl 10 g of tryptone 5 g of yeast extract Add deionized H2O to a final volume of 1 liter Adjust to pH 7.0 with 5 N NaOH Autoclave
LB Agar (per Liter)
10 g of NaCl 10 g of tryptone 5 g of yeast extract 20 g of agar Add deionized H2O to a final volume of 1 liter Adjust pH to 7.0 with 5 N NaOH Autoclave Pour into petri dishes (~25 ml/100-mm plate)
The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on the web at http://www.stratagene.com/MSDS/. Simply enter the catalog number to retrieve any associated MSDS??s in a print-ready format. MSDS documents are not included with product shipments.