GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. 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 O0 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 Remove the culture medium and add O0 5ml fresh warm complete medium Thaw the pre made lentiviral stock at room temperature and add the appropriate amount of virus stock to obtain the desired MOI Return cells to 37 C CO2 incubator Ready to use Lentiviral Particles for LacZ product manual Page 4 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 el Giae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com 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 each2. 7930 Arjons Drive Suite B San Diego CA 92126 el Grae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Pre made lentivirus for B Galactosidase expression Cat Product Name lacZ RFP Bsd lentiviral particles 1x10 IFU ml x 200ul LVP301 lacZ Puro lentiviral particles 1x10 IFU ml x 200ul lacZ Neo lentiviral particles 1x10 IFU ml x 200ul lacZ luciferase lentiviral particles 1x10 IFU ml x 200uI lacZ Bsd lentiviral particles 1x10 IFU ml x 200ul acZ GFP Bsd lentiviral particles 1x10 IFU ml x 200ul lacZ RFP puro lentiviral particles 1x10 IFU ml x 200ul LVPO21 GFP lacZ His Lentiviral particles 1x10 IFU ml x 200ul Storage lt 70 C avoid repeat freeze thaw cycles Stable for 6 months at lt 700C 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 RFP Bsd RFP Puro GFP Bsd on GFP Puro ee Op Puro Bsd Luciferase Pre made LacZ lentiviral particles are generated from GenTarget
3. integrated For inducible expression the tetR must be expressed in advance to stop the transcription and the expressed was activated by adding tetracycline This inducible expression is tetracycline s dose dependent In general the amount of tetracycline is used at 1 0 10 ug ml final concentration The image below illustrates how the inducible expression works Gentarget provides premade tetR particles with different antibiotics for double selecting the transduced inducible expression cells Ready to use Lentiviral Particles for LacZ product manual Page 2 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 el Giae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com suCMV 7 A a za gt 1 x 4 E E Tet operator gene tetR protein tet Expression particles Expression repressed tetR protein binds to tet operator sequences in promoter to repress the transcription if Add tet E Expression induced Tetracycline binds to tetR to release tetR from promoter and transcription is initiated ssion e Particles are used in a tetR expression stable cell line that constantly express tetR protein in advance e Transfect a tetR expression plasmid before transduce lentiviral particles e Co transduce both the tetR repressor particles and the gene expression particles into the sample cells with equal MOI and the double transdu
4. made lentivirus product category 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 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 Ready to use Lentiviral Particles for LacZ product manual Page 6 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 el Giae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Premde lentivirus for human and mouse iPS Myc NANOG iPS factors OCT4 SOX2 FLF4 factors with different fluorescent and antibitoic marke
5. s Optional inducible lentiviral system with different selection markers see vector Ready to use Lentiviral Particles for LacZ product manual Page 1 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 dil Giae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com core structure scheme above Full length LacZ ORF was fully verified by sequencing VSV G pseudotyped lentiviral particles are generated in 293T cell and provided in DMEM medium with 10 FBS 60ug m I of polybrene For more details about premade particles please see FAQ for pre made lentiviral particles pdf http www gentarget com pdf FAQ Premade Lentiviral particles pdf For product Cat LVPO21 the full length Beta galactosidase lacZ was fusioned with N term GFP protein and with C term a 6His under an inducible CMV promoter A RFP protein was bicistronically expressed under the same CMV promoter mediated via a F2A element see vector map scheme below So it has triple signals GFP RFP signals that can be visualized via microscope and lacZ signal via staining Please see the scheme for core lentivector structure Cat LVP021 F2A self cleavage site m 1 RFP PGK Bsd WPRE ZI 1 dsad About inducible expression LacZ was natively expressed without any tags under a tetracycline inducible suCMV promoter in which two tetracycline operator sequences was
6. ced cells can be selected by both antibiotics and then used for inducible expression Key features 1 2 ci High Lacz expression level and high viral titer Easy transduction monitoring via the RFP or GFP fluorescent signal under microscope not available for all particles Dual markers transduced cells can be sorted via a fluorescent signal or selected via antibiotics not available for all particles The lentivirus are ready and easy to use simply add 50ul into your cell culture in 24 well plate see sample image below Note dependent upon your specific needs you may design the transduction with different MOI for different levels of expression Ready to use Lentiviral Particles for LacZ product manual Page 3 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 el Giae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Transduction sample image Figure 1 50 ul of pre made lentiviral particles Cat LVP010 added into 293HEK cells At 72 hours after transduction image was taken under microscope with RFP filter Left image then cells washed with PBS and stained with lacZ staining kit for 10min take the bright light image to see lacZ stained cells Right image note prolonged staining time should show all lacZ positive cells which is not showed here Transduction Protocols 1 Transduction Protocol for Adhesive cells
7. 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 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 pos
8. itive cells and maintain antibiotic selection to generate a stable cell line Ready to use Lentiviral Particles for LacZ product manual Page 5 of 7 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 el raph nG Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Note Filter wavelength settings GFP filter Ex450 490 Em525 RFP filter Ex545 Em620 Safety Precaution Please use extra caution when using lentiviral particles Remember Ware glove all the time at handling Lentiviral particles Please refer CDC and NIH s links see references for more details regarding to safety issues References Molecular Therapy 2003 7 460 466 doi 10 1016 S1525 0016 03 00024 8 Annu Rev Microbiol 1994 48 345 69 Microbiol Mol Biol Rev 2005 Jun 69 2 326 56 NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors Link CDC guidelines for Lab Biosafety levels Link Ul BR WN eB Warranty This product is warranted to meet its quality as described when used 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
9. rs 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 treatment for lentiviruses validation of the specificity of any lentivirus target expression effects Ready to use lentivirus expressing specific enzymes with different selection markers expression Ready to use Lentiviral Particles for LacZ product manual Page 7 of 7 www gentarget com GenTarget Inc Copyrights 2015
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