10 µg lyophilized plasmid DNA (store lyophilized plasmid at 20 C)

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B IOLUMINESCENCE R ESONANCE E NERGY T RANSFER

R ENILLA L UCIFERASE F USION P ROTEIN E XPRESSION V ECTOR Product: pRluc-C Vectors

Catalog number: 6310210

Description: The pRluc-C vectors contain a multiple cloning site (MCS) located downstream of the Renilla Luciferase gene (Rluc) which acts as the Donor moiety in a BRET2 assay. The MCS allows for the subcloning of a gene of interest in order to create a fusion protein having the structure [Rluc:gene of interest]. The fusion protein gene is placed under the control of the cytomegalovirus (CMV) promoter thus assuring a very high constitutive expression in a variety of cells.

Amount: 10 µg lyophilized plasmid DNA (store lyophilized plasmid at –20°C)

Reconstitution Protocol

Reconstitution: • Centrifuge briefly to recover contents

• Reconstitute to 0.4 µg/µl with 25 µl of 10 mM Tris-HCl pH 8.0, 1 mM EDTA Storage conditions: • Store reconstituted plasmid at -20°C

• After thawing, centrifuge briefly to recover contents

Shelf life: • 1 year from date of receipt under recommended storage conditions

Quality Control Procedures

• The identity of the pRluc-C plasmids and the presence of the MCS restriction sites are confirmed by sequence analysis.

• The presence of RNA and chromosomal DNA as well as the proportion of superhelical DNA are determined by agarose gel electrophoresis using 1 µg of plasmid DNA.

• The absence of nuclease contamination is determined by agarose gel electrophoresis following incubation of 1 µg of plasmid DNA in standard restriction buffer for 16 hours at 37°C.

• The quantity and purity of DNA are determined by UV spectroscopy.

• The functionality of the plasmids is assessed by measuring luciferase activity upon transfection of CHO or BHK cells with LipofectAMINE. The intensity of the luciferase signal is compared to the signal of reference plasmids using a Fusion Universal Microplate Analyzer.

Renilla Luciferase Substrate

BRET2 requires a modified form of the Rluc coelenterazine substrate, called DeepBlueC. DeepBlueC has been selected for its ability to confer superior spectral properties to the reaction, resulting in excellent discrimination of the Rluc and GFP2 signals.

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pRluc-C1 Vector Map & Notes

Plasmid size: 4965 bp

Cloning sites: SacI, XhoI, EcoRI, PstI, EcoRV, HindIII, KpnI, SacII, XbaI (methylated)

Antibiotic resistance: Prokaryotic: Kanamycin (25 µg/ml for E.coli)

Eukaryotic: G418/Neomycin (concentration is cell type dependent)

PCMV 1 - 583

SV40 early poly (A) signal 1784 - 1834 Rluc gene 601 - 1533 TK poly (A) signal 3891 - 3954 Multiple cloning site (MCS) 1565 - 1641

PSV40/Pamp 2398 - 2812

Kan/NeoR 2861 - 3655

f1 origin 1884 - 2336 pUC sequences ( ori ) 4240 - 4883

pRluc-C1 Vector Multiple Cloning Site

pRluc-C1 Vector

GAA CAA CGG GCC CGG GAT CCC GGA CTC AGA TCT CGG CGA GCT CTC GAG

AAT TCT CAC GCG TCT GCA GGA TAT CAA GCT TGC GGT ACC GCG GGC CCG

GGA TCC ACC GGA TCT AGA TAA CTG ATC A

* Frame changes characterizing pRluc-C1, C2 and C3 vectors occur after this site.

Rluc BglII * SacI XhoI

EcoRl Pstl EcoRV HindIII KpnI

SacII

XbaI

Stop Stop Stop

Quality Control Data

• The identity of the pRluc-C1 plasmid and the presence of the MCS restriction sites have been confirmed by sequence analysis.

• Incubation in standard restriction enzyme buffer at 37°C for 16 hours showed no evidence of nuclease activity as detected by agarose gel electrophoresis.

• No RNA and chromosomal DNA were detected in a 1 µg sample of plasmid DNA following agarose gel electrophoresis.

• Percent DNA in Superhelical form: > 80%

• Purity (A260/A280) at pH 8.0: 1.75

• Transfection of CHO cells showed that the pRluc-C1 vector is functional and expressed Rluc levels within 25% of the corresponding reference plasmids.

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Plasmid size: 4966 bp

Cloning sites: SacI, XhoI, EcoRI, PstI, EcoRV, HindIII, KpnI, SacII, XbaI (methylated)

Antibiotic resistance: Prokaryotic: Kanamycin (25 µg/ml for E.coli)

Eukaryotic: G418/Neomycin (concentration is cell type dependent)

PCMV 1 - 583

SV40 early poly (A) signal 1785 - 1835 Rluc gene 601 - 1533 TK poly (A) signal 3892 - 3955 Multiple cloning site (MCS) 1566 - 1642

PSV40/Pamp 2399 - 2813

Kan/NeoR 2862 - 3656

f1 origin 1885 - 2337 pUC sequences ( ori ) 4241 - 4884

pRluc-C2 Vector Multiple Cloning Site

pRluc-C2 Vector

GAA CAA CGG GCC CGG GAT CCC GGA CTC AGA TCT CCG GCG AGC TCT CGA

GAA TTC TCA CGC GTC TGC AGG ATA TCA AGC TTG CGG TAC CGC GGG CCC

GGG ATC CAC CGG ATC TAG ATA ACT GAT CA

* Frame changes characterizing pRluc-C1, C2 and C3 vectors occur after this site.

Rluc BglII * SacI

XhoI

EcoRl Pstl EcoRV HindIII KpnI

SacII

XbaI

Stop Stop Stop

Quality Control Data

• The identity of the pRluc-C2 plasmid and the presence of the MCS restriction sites have been confirmed by sequence analysis.

• Incubation in standard restriction enzyme buffer at 37°C for 16 hours showed no evidence of nuclease activity as detected by agarose gel electrophoresis.

• No RNA and chromosomal DNA were detected in a 1 µg sample of plasmid DNA following agarose gel electrophoresis.

• Percent DNA in Superhelical form: > 80%

• Purity (A260/A280) at pH 8.0: 1.78

• Transfection of CHO cells showed that the pRluc-C2 vector is functional and expressed Rluc levels within 25% of the corresponding reference plasmids.

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pRluc-C3 Vector Map & Notes

Plasmid size: 4964 bp

Cloning sites: SacI, XhoI, EcoRI, PstI, EcoRV, HindIII, KpnI, SacII, XbaI (methylated)

Antibiotic resistance: Prokaryotic: Kanamycin (25 µg/ml for E.coli)

Eukaryotic: G418/Neomycin (concentration is cell type dependent)

PCMV 1 - 583

SV40 early poly (A) signal 1783 - 1833 Rluc (h) gene 601 - 1533 TK poly (A) signal 3890 - 3953 Multiple cloning site (MCS) 1564 - 1640

PSV40/Pamp 2397 - 2811

Kan/NeoR 2860 - 3654

f1 origin 1883 - 2335 pUC sequences ( ori ) 4239 - 4882

pRluc-C3 Vector Multiple Cloning Site

pRluc-C3 Vector

GAA CAA CGG GCC CGG GAT CCC GGA CTC AGA TCT GGC GAG CTC TCG AGA

ATT CTC ACG CGT CTG CAG GAT ATC AAG CTT GCG GTA CCG CGG GCC

CGG GAT CCA CCG GAT CTA GAT AAC TGA TC A

* Frame changes characterizing pRluc-C1, C2 and C3 vectors occur after this site.

Rluc BglII * SacI

XhoI

EcoRl Pstl EcoRV HindIII KpnI

SacII

XbaI

Stop Stop Stop

Quality Control Data

• The identity of the pRluc-C3 plasmid and the presence of the MCS restriction sites have been confirmed by sequence analysis.

• Incubation in standard restriction enzyme buffer at 37°C for 16 hours showed no evidence of nuclease activity as detected by agarose gel electrophoresis.

• No RNA and chromosomal DNA were detected in a 1 µg sample of plasmid DNA following agarose gel electrophoresis.

• Percent DNA in Superhelical form: > 80%

• Purity (A260/A280) at pH 8.0: 1.77

• Transfection of CHO cells showed that the pRluc-C3 vector is functional and expressed Rluc levels within 25% of the corresponding reference plasmids.

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BRET2TM is a trademark of Packard Instrument Company

DeepBlueC is a trademark of Packard Instrument Company. Patent pending

Renilla Luciferase is produced and sold under license, U.S. Patent Nos. 5,292,658 & 5,418,155, CA Patent No. 2,105,984, EPO Patent No. 91 908378.2, JP Patent No. 506544/91 exclusively licensed from Chemicon International, Inc.

Cytomegalovirus (CMV) is produced and sold under license, US Patent Nos 5,1168,062 and 5,385,839 LipofectAMINE is a trademark of Life Technologies, Inc.

Fusion is a trademark of Packard Instrument Company.

LIMITED WARRANTY BioSignal Packard warrants that, at the time of shipment, the products sold by it are free from defects in material and workmanship and conform to specifications which accompany the product. BioSignal Packard makes no other warranty, express or implied, with respect to the products, including without limitation any warranty of merchantability or fitness for any particular purpose. Notification of any breach of warranty must be made within 30 days of receipt unless provided in writing by BioSignal Packard. No claim shall be honored if the customer fails to notify BioSignal Packard within the period specified. The sole and exclusive remedy of the customer for any liability of BioSignal Packard of any kind, including liability based upon warranty (express or implied whether contained herein or elsewhere), strict liability, breach of contract or otherwise, is limited, at BioSignal Packard's sole election, to replacement of the goods or a refund of the invoice price of the goods. BioSignal Packard shall not in any case be liable for special, incidental or consequential damages of any kind.

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