GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1.2. 5 Easy selection via Fluorescent signal or antibiotic marker Transduction Protocols 1 Transduction Protocol for Adhesive cells Note Pre made lentivirus is provided ready to use so it can be simply added to your cell culture the amount of virus to add depends on the cell type For quick transduction add 50 ul of virus into each well of 24 well plate where cell density is 5096 to 7596 After 72 hours no need to change medium visualize positive transduction rate by fluorescence microscopy For stable cell line generation passage cells into medium containing antibiotic or perform fluorescence cell sorting followed by antibiotic selection Day 0 Seed cells in complete medium at the appropriate density and incubate overnight Note at the time of transduction cells should be 5096 7596 confluent For example seed HeLa cells at 0 5 x 10 ml x 0 5ml in a well of a 24 well plate AMSBIO www amsbio com infoeamsbio com e UK amp Rest of the World BE North America Germany Switzerland aS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 449 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 Day 1 e Remove the culture medium and add 0 5
3. AMSBIO www amsbio com infoeamsbio com e UK amp Rest of the World BE North America Germany Switzerland 74953 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 449 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 Warranty This product is for research use only It is warranted to meet its quality as described when used accordance with its instructions Amsbio disclaims any implied warranty of this product for particular application In no event shall Amsbio be liable for any incidental or consequential damages in connection with the products Amsbio s sole remedy for breach of this warranty should be at Amsbio s option to replace the products AMSBIO www amsbio com infoeamsbio com AUZ UK amp Rest of the World BE North America Germany Switzerland ESS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218
4. Pre made Lentiviral Particles for Fluorescent Target fusion proteins Catalogi Product Name Amounts Luci P LVP673 Ener ure 1x10 IFU ml x 200ul fusion lentiviral particles Luci P LVP674 NOE Pure 1x10 IFU ml x 200ul fusion lentiviral particles Luci P LVP675 EM Tus une 1x10 E x 200ul fusion lentiviral particles N LVP676 GEP Hienersse NED 1x10 1x10 IFU mix 20041 x 200ul fusion lentiviral particles RFP Luciferase Neo LVP677 fusion lentiviral particles 1x10 IFU ml x 200ul LVP442 ute dio LN 1x10 IFU ml x 200ul control lentiviral particles LVP443 niis dio NNNM 1x10 IFU ml x 200ul control lentiviral particles LVP399 R een 1x10 IFU ml x 200ul lentiviral particles LVP399 G pn 1x10 IFU ml x 200ul lentiviral particles LVP399 C Und 1x10 IFU ml x 200ul lentiviral particles m viral GFP Histone 2B fusion lentivira 1x10 IFU ml x 200ul BEEN particles m son lentiviral RFP Histone 2B fusion lentivira 1x10 IFU ml x 200ul particles P WEN CFP Histone 2B fusion lentivira 1x107 IFU ml x 200ul particles LVPA445 G ere omen een 1x10 IFU ml x 200ul Lentiviral particles LVP445 R nuns cd 1x10 IFU ml x 200ul Lentiviral particles LVP445 C San unn 1x10 IFU ml x 200ul Lentiviral particles LVPA446 G aE pcan on 1x10 IFU ml x 200ul Lentiviral particles LVP446 R ee dine 1x10 IFU ml x 200ul Lentiviral particles AMSBIO www amsbio com infoeamsbio com z4 UK amp Rest of the World BE North Am
5. cell types or organs including stem cells primary cells and non dividing cells both in vivo and in cell culture system Particles stably integrate into the transduced cell s genome for long term expression Therefore lentivirus holds unique promise as a gene transfer agent Pre made lentiviral particles expressing GFP RFP CFP Target fusion constructs are generated from Amsbio s high expression lentiviral system A fluorescent protein GFP RFP or CFP is cloned in frame with a target such as human or mouse ORF expressed under a proprietary suCMV promoter that demonstrates the highest expression levels 3 10 fold higher than CMV promoter in pCDNA6 3 vector cell type dependent Each fluorescent protein is codon optimized to generate brighter fluorescent signal They are great tools for sub cellular pathway studies in vivo signal transduction research living cell imaging protein interaction studies and many other applications The positive transduced cells can be sorted via the fluorescent signal or via Puromycin antibiotic selection See vector scheme below for vector structures Scheme s of Fluorescent Taraet fusion lentivectors ZIIIFPZ 93 j 4 T m I fusion target AMSBIO www amsbio com infoeamsbio com gt S UK amp Rest of the World BEES North America Germany rv Switzerland ZAS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E
6. Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 These Lentiviral Particles are great tool for e Sub cellular pathway studies e in vivo signal transduction research e Live cell imaging protein interaction studies and many other applications The positively transduced cells can be sorted by the fluorescent signal or selected for puromycin neomycin blasticidin resistance depending on the product marker selection type Key features 1 High titer and robust Amsbio s premade lentiviral particles are best in the class demonstrating the brightest fluorescent signal and strong transduction efficiency All particles are validated on lot by lot basis and their quality is guaranteed 2 Delivery of fluorescent labeled targets into hard to transfect cell lines like primary cells or neuron cells for long term expression 3 Very easy to use Particles are provided in DMEM medium with 1096 FBS and 60ug ml polybrene for ready to use status Simply add into your cell culture and visualize the fluorescence after 48 92 hours No need for any additives components or medium changes 4 Different fluorescent labeled particles for multi color application when using multiple different particles in same cells
7. 10 IFU ml x 200ul ME Lentiviral particles LVP550 C BEP EIUS 1x10 IFU ml x 200ul KNEE Lentiviral particles LVP551 C E KONNE EN 1x10 IFU ml x 200ul MEE Lentiviral particles LVP552 C CEP TREVA USON 1x10 IFU ml x 200ul cS Lentiviral particles LVP554 C pat Bee en 1x10 IFU ml x 200ul Lentiviral particles LVP556 C ae een 1x10 IFU ml x 200ul I NS Lentiviral particles NuEG GFP Null fusion control 1x10 IFU ml x 200ul AMSBIO www amsbio com infoeamsbio com z4 UK amp Rest of the World BE North America Germany Switzerland aS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 amsbio Nul R RFP Null fusion control 1x10 IFU ml x 200ul CFP Null fusion control 1x10 IFU ml x 200ul Storage lt 70 C avoid repeat freeze thaw cycles Stable for gt 6 months Product Description Lentiviral system is a gene delivery tool that uses lentivectors for gene expression or knockdown Lentivectors are HIV 1 Human Immunodeficiency Virus 1 derived plasmids used to generate lentiviral particles lentivirus that can be transduced into all kind of mammalian
8. add an equal amount of fresh medium containing relevant antibiotics Note amount of antibiotic depends on cell type Continue growing cells in CO incubator AMSBIO www amsbio com info amsbio com e UK amp Rest of the World BE North America Germany r3 Switzerland ESS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 449 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 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 Quick transduction examples LVP442 50ul LVP447 R LVP448 C GFP filter RFP filter CFP filter Add 50ul each lentivirus into one well in 24 well plate where cell density is at 50 75 in different cell types HEK293 A549 PC3 from left to right Images taken 72 hours after virus added no medium changed Result gt 90 of positive transduced cells Note Filter wavelength settings GFP filter Ex450 490 Em525 RFP filter Ex545 Em620 CFP filter Ex436 Em480 Attachment Pre made lentivirus pr
9. erica Germany Switzerland aS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 49 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 7 i f i LVP446 C eene 1x10 IFU ml x 200ul Lentiviral particles LVP447 G Chr TAT meen 1x10 IFU ml x 200ul Lentiviral particles LVP447 R peel 1x10 IFU ml x 200ul ME Lentiviral particles LVP447 C pee UR 1x10 IFU ml x 200ul MEE Lentiviral particles LVP448 G SES UID 1x10 IFU ml x 200ul Lentiviral particles LVP448 R Ree APSR USON 1x10 IFU ml x 200ul Lentiviral particles LVP448 C CrP PaA ON 1x10 IFU ml x 200ul GE RE Lentiviral particles LVP449 G ie 1x10 IFU ml x 200ul Ic IIS Lentiviral particles LVP449 R RESO AUSIN 1x10 IFU ml x 200ul BEEN Lentiviral particles LVP449 C ERES USE 1x10 IFU ml x 200ul MEE Lentiviral particles LVP550 R RI E CLCNZ RION 1x10 IFU ml x 200ul CREER Lentiviral particles LVP551 R Bee cle SIR 1x10 IFU ml x 200ul a Lentiviral particles LVP552 R Bee Reason 1x10 IFU ml x 200ul Lentiviral particles LVP554 R Ber IREE tusio 1x10 IFU ml x 200ul Lentiviral particles LVP556 R Bee CSPE TUSIGN 1x10 IFU ml x 200ul Lentiviral particles LVP556 G APAA 1x
10. g Express full length B galactosidase lacZ with different selection markers Pre made shRNA Premade shRNA lentivirus for knockdown a specific genes P53 LacZ Luciferase lentivirus and more microRNA and Premade lentivirus expression human or mouse precursor miRNA and anti miRNA anti microRNA lentivector and virus for human and mouse miRNA lentivirus Negative control Premade negative control lentivirus with different markers serves as negative lentiviruses control for lentivurs treatment for validation of the specificity of lentivirus target expression effect Other Enzyme Ready to use lentivirus expressing a specific enzyme with different selection expression markers Safety Precaution Amsbio lentiviral particles have adopted the most 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 Il cabinet Ware gloves at all times when handling lentiviral particles Please refer to CDC and NIH s guidelines for more details regarding the safety issues References 1 JVirol 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 5 CDC guidelines for Lab Biosafety levels link
11. ml 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 MOI e Return cells to 37 C CO incubator Note Try to avoid freeze thaw cycles If you do not use all of the virus at once 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 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 measure viability should be 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 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 CO incubator Day 2 At 24 hours after transduction
12. oducts Product Product Description Category please Category name to see product s pages protein Human and Premade lentivirus expressing human and mouse ORFs with RFP Blasticidin fusion AMSBIO www amsbio com info amsbio com SS UK amp Rest of the World BE North America Germany Switzerland aS 184 Park Drive Milton Park 1035 Cambridge Street Bockenheimer Landstr 17 19 Centro Nord Sud 2E Abingdon OX14 4SE UK Cambridge MA 02141 60325 Frankfurt Main CH 6934 Bioggio Lugano T 44 0 1235 828 200 T 1 617 945 5033 or T 49 0 69 779099 T 41 0 91 604 55 22 F 44 0 1235 820 482 T 1 800 987 0985 F 449 0 69 13376880 F 41 0 91 605 17 85 F 1 617 945 8218 amsbio Luciferase Premade lentivirus for luciferase protein expression firefly and Renilla with different antibiotic selection markers CRE Premade lentivirus for expressing nuclear permeant CRE recombinase with LoxP Premade lentivirus expressing LoxP GFP Stop LoxP RFP cassette used to monitor the CRE recombination event in vivo TetR inducible Premade lentivirus expressing TetR tetracycline regulator protein the repressor expression protein for the inducible expression system repressor E A iPS factors factors with different fluorescent and antibiotic markers Expression Cell Organelle Premade lentivirus for cell organelle imaging The fluorescent marker imaging GFP RFP CFP is localized in different cell organelles for living cell imagin
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