GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. 7930 Arjons Drive Suite B San Diego CA 92126 ai quae nG Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Pre made Lentiviral Particles for intracelular labeling LocLight Living cell imaging lentivirus for sub cellular localization GenTarget LocLight cell organelle labeling lentivirus was provided as 200ul per vial with different fluorescent markers at the titer of 1x10 IFU mL Please see the following table for product information LocLight Targeted Sub cellular Product Catalog Particles Organelle Nuc GFP Fluorescent signal only showed in Nuc RFP nucleus area targeted by an improved LVP360 LVP360 LVP360 Nuc CFP NLS Nuclear localization sequence G R C signal Particles contains Puromycin LVP450 LVP450 C marker Cyto GFP Fluorescent signal only showed in Cyto RFP Cytoplasm area targeted by an LVP450 LVP450 Cyto CFP engineered nuclear export signal _ Particles contains Puromycin marker LVP606 LVP606 R C LVP451 LVP451 R C QG R Fluorescent signal targeted in Endoplasmic Reticulum ER by the C l LVP606 terminal KDEL ER retention signal R Particles contains Puromycin marker Golgi GFP Fluorescent signal targeted in Golgi Golgi RFP by the golgi retention signal from 1 4 LVP451 Golgi CFP galactosyltransferase GT Particles R contains Blasticidin marker LVP452 LVP452 R C LVP453 LVP453 R C G G G Mito GFP Fluoresce2. Product Description Category please click category name to see product s pages Human and Premade lentivirus expressin a human mouse or rat gene with mouse ORFs RFP Blastididin fusion dual markers Fluorescent Preamde lentivirus express human codon optimized fluorescent markers protein GFP RFP CFP BFP YFP 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 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 Ready to use lentiviral Particles LocLight Living cell imaging lentivirus product manual Page 7 of 8 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 e quae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget
3. seed HeLa cells at 0 5 x 10 ml x 0 5ml in a well of a 24 well plate Day 1 e Remove the culture medium and add 0 5ml fresh warm complete medium e Thaw the pre made lentiviral stock at room temperature and add the appropriate amount of virus stock to obtain the desired MOT e 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 e Thaw lentiviral particles at room temperature Ready to use lentiviral Particles LocLight Living cell imaging lentivirus product manual Page 5 of 8 www gentarget
4. LVP444 DE n DA ai H2B Particles contains Puromycin a marker Lysosomes GFP Fluorescent signal targeted in Lysosomes RFP Lysosomes by lysosomal associated LVP457 LVP457 LVP457 Lysosomes CFP membrane protein 1 LAMP1 Particles contains Blasticidin marker LVP458 C contains Puromycin marker 4 i E i Endosomes GFP Fluorescent signal targeted in Endosomes RFP Endosomes by RAB5A that localized to Endosomes CFP early endosomes for endocytosis and cd ae endocytic sorting pathways Particles GFP Null control Fluorescent marker fusioned with a RFP Null control non targeting sequence Null serves Null G Null R Null C Puro Puro Puro CFP Null control 3s controls with fluorescent signal evenly distributed in entire cell Particles contains Puromycin marker Storage lt 70 C avoid repeat freeze thaw cycles Stable for gt 6 months Ready to use lentiviral Particles LocLight Living cell imaging lentivirus product manual Page 2 of 8 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 e arae hic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Product Description Lentivirus is the easiest and most effective method for delivering genes into the majority of mammalian cell types including non dividing and primary cells It allows genes to be integrated into the host cell geno
5. com __ antibitoic markers T antigen Express different large and small T antigen with different selection Expression markers 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 treatment for lentiviruses validation of the specificity of any lentivirus target expression effects Other Ready to use lentivirus expressing a specific enzymes with Enzyme different selection markers expression Ready to use lentiviral Particles LocLight Living cell imaging lentivirus product manual Page 8 of 8 www gentarget com GenTarget Inc Copyrights 2015
6. com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 ai arae hic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com e 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 e 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 depends on cell type Continue growing cells in CO2 incubator Day 3 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 Transduction example images 50 ul of each particles was added into HEK293 cells in one well at 24 well plate Image was taken at 48 hours after virus addition Note Filter wavelength settings CFP filter Ex436 Em480 GFP filter Ex450 490 Em525 RFP filter Ex545 Em620 BFP filter Ex380 Em460 Cyto GFP Cyto RFP Nuc GFP Nuc RFP CATH LVP450 G CATHLVP450 R CATHLVP360 G CAT LVP360 R gt AG Cyto GFP Nuc RFP GFP H2B MUG MEMBISne SEE skootah CAT LVP450 G CATHLVP440 G CAT LVP453 G i LVP360 R Ready to use lentiviral Par
7. der the control of the RSV promoter see the vector core structure scheme above which provides an easy way to select positive cells for long term research Ready to use lentiviral Particles LocLight Living cell imaging lentivirus product manual Page 3 of 8 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 ai arue ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Key features e Robust Expression and High Titer GenTarget s Premade Lentiviral Particles have the brightest fluorescence and the strongest transduction efficiency of any lentiviral particles on the market e Long Term Expression GenTarget s Premade Lentiviral Particles produce long lasting expression of fluorescently labeled target proteins even in hard to transfect cell lines such as primary and neuronal cells e Easy Transduction Simply add the Particles to your cell culture and visualize fluorescence in 48 92 hours There is no need for any additives or changes of medium e Multiple Colors Particles expressing different colors may be transduced into the same cells for multi color applications e Easy Selection of Transduced Cells Use either fluorescence signal or puromycin resistance e Tested and Validated Each lot of Particles is validated and guaranteed to be of the highest quality e Ready to Use Simply add virus into your cell culture For general questions about our rea
8. dy to use lentiviral particles please See FAQs for pre made _ lentiviral particles pdf on our website http www gentarget com wp content uploads 2013 07 FAQ Premade Lentiviral particles pdf GenTarget provides lentiviral services for cloning your gene of interest and generating ready to use viral particles with the best prices and fastest turnaround times Please see our website for details 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 Ready to use lentiviral Particles LocLight Living cell imaging lentivirus product manual Page 4 of 8 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 e arae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Day 0 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
9. me for long term expression Utilizing our proprietary lentiviral vector systems GenTarget has generated the LocLight Sub Cellular Labeling Lentivirus product line These premade lentiviruses contain a well defined organelle targeting signal fusioned to a fluorescent protein GFP RFP or CFP and expressed under our proprietary suCMV promoter A non targeting spacer sequence Null was fusioned with fluorescent which serves as the control lentivirus The Null control virus demonstrate a evenly distributed fluorescent signal inside the cell The lentivirus also contains an antibiotic marker Puromycin Neomycin or Blasticidin under a Rsv promoter see lentivector s core structure scheme below Core expression cassette of LocLight particles Sub cellular localization element The fluorescent proteins are non toxic to cells do not compromise cell structure or interfere with signaling pathways They are therefore ideal tools for live cell imaging and dynamic investigation of sub cellular signal pathways Pre made LocLight sub cellular labeling lentiviruses are extremely easy to use and do not require any additives or substrates Simply add the lentivirus into a mammalian cell culture The expression of auto fluorescent protein will be localized to the specific sub cellular compartments at 1 3 days post transduction and can be visualized by fluorescence microscopy Each particle also contains a puromycin antibiotic selection marker un
10. nt signal targeted in Mito RFP Mitochondria by the Leader sequence LVP452 Mito CFP of E1 alpha pyruvate dehydrogenase G Particles contains Blasticidin marker G Ready to use lentiviral Particles LocLight Living cell imaging lentivirus product manual Page 1 of 8 Nuc membrane Fluorescent signal targeted in GFP Nuclear Membrane by the inner Nuc membrane nuclear membrane localization signal LVP453 RFP from lamin B membrane receptor Moe memoran Particles contains Puromycin marker www gentarget com GenTarget Inc Copyrights 2015 CFP 7930 Arjons Drive Suite B San Diego CA 92126 e arae lic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Peroxisome GFP Fluorescent signal targeted in Peroxisome RFP Peroxisome by the Peroxisomal C Peroxisome CFP terminal SKL targeting sequence LVP454 LVP454 LVP454 i i R C Particles contains Puromycin marker Plasma mem GFP Fluorescent signal targeted in Plasma mem RFP Plasma membrane by ADP GDA i R membrane protein Particles contains Puromycin marker G G Microtubule GFP Fluorescent signal targeted in Microtubule RFP Microtubule by microtubule LVP456 LVP456 LVP456 Microtubule CFP associated protein 4 MAPA Particles G R C G G G contains Puromycin marker GFP H2B Fluorescent signal targeted in RFP H2B mitotic chromosomes and interphase CFP H2B chromatin by Histone 2B protein
11. ticles LocLight Living cell imaging lentivirus product manual Page 6 of 8 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 e arae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com 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 Wear glove all the time when handling Lentiviral particles Please refer CDC and NIH s guidelines for more details regarding to safety issues References 1 J Virol 2000 November 74 22 10778 10784 2 Hum Gene Ther 2003 14 1089 105 3 Mol Ther 2002 6 162 8 4 NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors Link Warranty This product is for research use only It is warranted to meet its 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
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