GFP-lacZ (His) - GenTarget Inc
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1. 4 The lentivirus are ready and easy to use simply add 50ul into your cell culture Furin 2A self cleavage site O T I RFP PGK Bsd WPRE J n Q 7 ha D O N Transduction Protocols 1 Transduction Protocol for Adhesive cells Note Pre made lentivirus is provided ready to use so it can be simply added into your cell culture the amount of virus to add depends on cell type For quick transduction add 50 ul of virus into each well of 24 well plate where cell density is 50 to 75 After 72 hours no need to change medium visualize positive transduction rate by fluorescence microscopy For stable cell line generation pass cells into medium containing antibiotic or perform fluorescence cell sorting followed by antibiotic selection Day O Seed cells in complete medium at the appropriate density and incubate overnight Note at the time of transduction cells should be 50 75 confluent For example seed HeLa cells at 0 5 x 10 ml x 0 5ml in a well of a 24 well plate Day 1 Pre made Lentiviral Particles for GFP_lacZ fusion product manual Page 2 of 5 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 el aruel ne Phone 858 6788683 Fax 800 3804198 orders gentarget com Remove the culture medium and add 0 5ml fresh warm complete medium Thaw the pre made lentiviral stock at room temperature and add the appropriate amount of viru2. RFP ColorSwitch cassette used to monitor the CRE recombination event in vivo CRISPR hu Preamde lentivirus express humanzied wild type Cas9 CAS9 endonuclease for genomic editing with CRISPR TetR Premade lentivirus expressin TetR tetracycline regulator protein inducible the repressor protein for the inducible expression system expression repressor Premde lentivirus for human and mouse iPS Myc NANOG iPS factors OCT4 SOX2 FLF4 factors with different fluorescent and antibitoic markers T antigen Express SV40 large T antigen with different selection markers Expression Cell Premade lentivirus for cell organelle imaging The fluorescent Organelle marker GFP RFP CFP was sub cellular localized in different imaging cell organelle for living cell imaging LacZ Express different full length B galactosidase lacZ with different expression selection markers Anti miNA Pre made lentivirus expression a specific anti miRNA cassette lentivirus Fluorescent Pre made lentivirus expression a GFP RFP CFP ORF fusion ORF fusion target Pre made Premade shRNA lentivirus for knockdown a specific genes P53 shRNA LacZ Luciferase and more lentivirus microRNA Premade lentivirus expression human or mouse precursor and anti miRNA And anti miRNA lentivector and virus for human and microRNA mouse miRNA lentivirus Negative Premade negative control lentivirus with different markers control serves as the negative control of lentivurs tr
3. G 7930 Arjons Drive Suite B San Diego CA 92126 Genaro nec Phones 8 6788683 Fax 800 3804198 orders gentarget com Pre made Lentiviral Particles for GFP_lacZ fusion Cat Product Name Amounts GFP lacZ his particles 200ul 1 x 10 IFU mL in LVPO21 DMEM containing 10 FBS and 60ug ml polybrene LVP021 PBS GFP lacZ his particles 200ul 5 x 10 IFU mL in PBS E In vivo ready solution Storage lt 70 C avoid repeat freeze thaw cycles Stable for 6 months at lt 70 C Product Description GenTarget s lentivector system is Human Immunodeficiency Virus 1 HIV based plasmids for gene expression and knockdown The lentivectors are used to generate lentiviral particles lentivirus that can be transduced into almost all kinds of mammalian cells including stem cells primary cells and non dividing cells both in vivo and in vitro Lentiviral Particles stably integrate into the transduced cells genome for long term expression making it a great gene transfer agent Pre made GFP_lacZ fusion Lentiviral particles are generated from GenTarget s SureTiter lentiviral system expressing GFP and the full length Beta galactosidase lacZ fusion gene under suCMV promoter Both GFP and lacZ are fully functional GFP LacZ fusion was expressed with C term His tag A RFP was bibicistronically expressed under the same suCMV promoter for titer monitoring So they are triple signal particles with GFP RFP sig
4. eatment for lentiviruses validation of the specificity of any lentivirus target expression effects Other Ready to use lentivirus expressing specific enzymes with Enzyme different selection markers expression Pre made Lentiviral Particles for GFP_lacZ fusion product manual Page 5 of 5 www gentarget com GenTarget Inc Copyrights 2015
5. nals that can be visualized via microscope and with lacZ signal via staining A blasticidin gene under PGK promoter allows to select stable cells for long term expression VSV G pseudotyped lentiviral particles are generated in 293T cell The particles are provided in two formats e packaged in 10 of FBS in DMEM containing 10 FBS and 60ug ml_ of polybrene Cat LVP021 e packaged in PBS solution Cat LVP021 PBS The particles in PBS solution are best suitable for serum free cultures or for hard to transduced cell types For more details about premade particles please see FAQ for pre made lentiviral particles pdf Pre made Lentiviral Particles for GFP_lacZ fusion product manual Page 1 of 5 www gentarget com GenTarget Inc Copyrights 2015 G 7930 Arjons Drive Suite B San Diego CA 92126 Gent ayaa ne Phones 8 6788683 Fax 800 3804198 orders gentarget com Key features 1 Lentiviral particles contain RFP blasticidin resistant gene allowing to generate GFP lacZ fusion stable cell lines by Blasticidin antibiotic selection or via fluorescent cell sorting It is actually triple signals GFP LacZ and RFP particles 2 GFP fusioned with full length LacZ B Galactosidase was expressed with a C term His tag 3 The strongest suCMV promoter make the pre made virus a ideal tool for mammalian protein expression stable cell line construction and enzymatic assays both in vivo or in vitro see schematic vector map below
6. pends on cell type Continue growing cells in CO2 incubator Day 3 Pre made Lentiviral Particles for GFP_lacZ fusion product manual Page 3 of 5 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 el aruel ne Phone 858 6788683 Fax 800 3804198 orders gentarget com At 72 hours after transduction check fluorescence with a fluorescence microscope or calculate the transduction efficiency using a cell sorter such as FACS or Guava Sort for fluorescence positive cells and maintain antibiotic selection to generate a stable cell line Note Filter wavelength settings GFP filter Ex450 490 Em525 RFP filter Ex545 Em620 Safety Precaution Gentarget lentiviral particles adapts must advanced lentiviral safety features using the third generation vectors with self inactivation SIN 3UTR and the premade lentivirus is replication incompetent However please use extra caution when using lentiviral particles Use the lentiviral particles in Bio safety II cabinet Ware glove all the time at handling Lentiviral particles Please refer CDC and NIH s guidelines for more details regarding to safety issues References 1 NIH stem cell training program Link 2 NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors Link 3 CDC guidelines for Lab Biosafety levels Link Warranty This product is for research use only It is warranted to meet its
7. quality as described when used in accordance with its instructions GenTarget disclaims any implied warranty of this product for particular application In no event shall GenTarget be liable for any incidental or consequential damages in connection with the products GenTarget s sole remedy for breach of this warranty should be at GenTarget s option to replace the products Related Products GenTarget s pre made lentivirus product category Product Product Description Category please click category name to see product s pages Human Premade lentivirus expressin a human mouse or rat gene with mouse or rat RFP Blastididin fusion dual markers ORFs Fluorescent Preamde lentivirus express human codon optimized fluorescent markers protein GFP RFP CFP BFP YFP Pre made Lentiviral Particles for GFP_lacZ fusion product manual Page 4 of 5 www gentarget com GenTarget Inc Copyrights 2015 GeniTarset inc 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Fax 800 3804198 orders gentarget com Luciferase Premade lentivirus for all kinds of luciferase protein expression expression firefly and Renilla with different antibiotic selection markers CRE Premade lentivirus for expressing nuclear permeant CRE recombinase recombinase with different flurescent and antibiotic markers LoxP Premade lentivirus expressing LoxP GFP Stop LoxP
8. s stock to obtain the desired MOI Return cells to 37 C CO2 incubator Note Try to avoid freezing and thawing If you do not use all of the virus at one time you may re freeze the virus at 80 C for future use virus titer will decrease by 10 for each freeze thaw cycle Day 3 At 72hr after transduction check the transduction rate by fluorescence microscopy or calculate the exact transduction rate by flow cytometry FACS or Guava Day 3 optional Sort transduced cells by FACS and select for antibiotic resistance A pilot experiment should be done to determine the antibiotic s kill curve for your specific cell line refer to the pertinent literature on generation of stable cell lines 2 Transduction Protocol for Suspension Cells Grow cells in complete suspension culture medium use a shaking flask in a CO incubator if necessary Measure cell density When density has reached 3 x 10 cells ml measured viability should be gt 90 Dilute cells into 1 x 10 cell ml in complete medium Day 1 Thaw lentiviral particles at room temperature Add premade lentiviral particles into the diluted cells at a ratio of 50 to 100 ul virus per 0 5 ml of cells Note depending on cell type you may need to use more or less virus Grow cells in a shaking flask in a CO2 incubator Day 2 At 24 hours after transduction add an equal amount of fresh medium containing relevant antibiotics Note amount of antibiotic de
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