GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. 7930 Arjons Drive Suite B San Diego CA 92126 ai quae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Pre made reporting lentivirus for Wnt signaling activity _Cat ProductName Amounts LVP808 P or Wnt Tcf GFP Puro LVP808 P PBS Lentiviral ee LVP809 P P PBS iad partes LVP810 P or Wnt Tcf Luc Puro LVP811 P PBS Lentiviral particles LVP808 B PBS Lentiviral particles LVP809 B PBS Lentiviral particles LVP810 B or Wnt Tef Luc N 7 LVP811 B B PBS L ntiviral baei Wnt Tcf GFP Neo Or LVP808 N N PBS Lentiviral 1 cs Wnt Tcf RFP Neo 200ul 1 x 108 IFU mL in LVP809 N PBS Lentiviral particles LVP810 Nor Wnt Tcf Luc Neo TORANN LVP810 N PBS Lentiviral particles LVP811 N or Wnt Tcf Rluc Neo LVP811 N PBS Lentiviral particles LVP808 R PBS Lentiviral particles LVP810 R PBS Lentiviral particles LVP811 R PBS Lentiviral particles LVP809 G PBS Lentiviral particles LVP810 G PBS Lentiviral particles LVP811 G PBS Lentiviral particles Storage _ lt 70 C avoid repeat freeze thaw cycles Stable for gt 6 months Product Description Lentiviral system is a gene delivery tool using lentivectors for gene expression or knockdown 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 Ready to use Lentiviral Particles for Wnt si2. free bacterial and mycoplasma contamination Virus titers were tested lot by lot The lentivirus are ready and easy to use simply add 50ul into one well of your cell culture in 24 well plate and select or sort the positive transduced cells at 2 3 days post virus transduction Or simply go for wnt signal induction without the selection The readout can be easily monitored by luciferase assay or via the Fluorescent microscope or F readers depending on product report type Ready to use luciferase lentiviral particles are provided in two formats 1 2 Packaged in 10 of FBS in DMEM containing 10 FBS and 60ug mi of polybrene 10x Particles were concentrated and buffer exchanged in PBS without any human or animal origin components The virus in PBS is good for any cell types that requires non serum in the medium or good for hard to infect cell types For more details about premade particles please see FAQ for pre made lentiviral particles pdf 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 med
3. RFP Blastididin fusion dual markers ORFs Fluorescent Preamde lentivirus express human codon optimized fluorescent markers protein GFP RFP CFP BFP YFP Ready to use Lentiviral Particles for Wnt signaling product manual Page 5 of 6 www gentarget com GenTarget Inc copyright 2015 Luciferase expression LoxP ColorSwitch CRISPR hu CAS9 TetR inducible expression repressor iPS factors T antigen Express SV40 large T antigen with different selection markers Expression Premade lentivirus for cell organelle imaging The fluorescent marker GFP RFP CFP was sub cellular localized in different Cell Organelle imaging Lacz Express different full length B galactosidase lacZ with different expression selection markers Pre made lentivirus expression a specific anti miRNA cassette Pre made lentivirus expression a GFP RFP CFP ORF fusion target Premade shRNA lentivirus for knockdown a specific genes P53 Anti miNA lentivirus Fluorescent ORF fusion Pre made shRNA lentivirus microRNA and anti microRNA lentivirus Negative control lentiviruses Other Enzyme expression Ready to use Lentiviral Particles for Wnt signaling product manual Page 6 of 6 GenTarget Inc GenTaruet Ine Premade lentivirus for all kinds of luciferase protein expression firefly and Renilla with different antibiotic selection markers CRE Premade lentivirus for expressing nuclear
4. ed 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 PLoS One Vol 5 No 2 2010 2 The ins and outs of Wingless signaling Trends Cell Biol 2004 Jan 14 1 45 53 3 Wnt genes Cell 1992 69 7 1073 1087 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 GentTarget s option to replace the products Note Filter wavelength settings BFP filter Ex380 Em460 CFP filter Ex436 Em480 GFP filter Ex450 490 Em525 YFP filter Ex500 Em535 RFP filter Ex545 Em620 Related Products GenTarget s pre 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
5. ein is present the luminescent or fluorescent report is expressed which can be easily readout via luciferase assay or by fluorescent microscope Those reporting lentivirus also contains a different fluorescent selection marker or an antibiotic selection marker under a constitutive RSV promoter which make it easier to select the stably infected Wnt signal reporting cells like generate WNT sensor cell lines Those reporting lentivirus products provide the efficient and easy tools to probe and manipulate Wnt signaling Premade Wnt signal reporting lentivirus The ready to use lentivirus expresses a report firefly Luciferases Renilla luciferase GFP or RFP under Wnt responsive promoter The report is only expressed when the Wnt stimulating signal is present Each lentivirus product contains a constitutively expressed selectable marker Fluorescent or antibiotic under a separated RSV promoter See the lentivector core cassette in below scheme Ready to use Lentiviral Particles for Wnt signaling product manual Page 2 of 6 www gentarget com GenTarget Inc copyright 2015 7930 Arjons Drive Suite B San Diego CA 92126 ai quae nG Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Lentivector core scheme Wnt Signal pathway Bsd 8 x Tcf RSV __ Puro TRE promoter Neo RFP GFP Lentivirus was pseudotyped with VSVG envelope protein produced in 293T cells All particles were tested to be
6. gnaling product manual Page 1 of 6 www gentarget com GenTarget Inc copyright 2015 7930 Arjons Drive Suite B San Diego CA 92126 e arae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com lentivirus that can be transduced into almost all kinds of mammalian cells including stem cells primary cells and non dividing cells both jn vivo and in vitro Lentiviral Particles stably integrate into the transduced cells genome for long term expression making it a great gene transfer agent The Wnt signaling pathways are a group of signal transduction pathways made of proteins that pass signals from outside of a cell through cell surface receptors to the inside of the cell Wnt signaling pathways are activated by the binding of a Wnt protein a large family of secreted glycoproteins such as wnt3A Wnt1 and so on to a family receptor The Wnt signaling pathway plays important roles in cell cell proliferation differentiation and survival and many other cell developing aspects GenTarget developed a set of reporting lentivirus for monitoring or manipulating the Wnt pathway s activity in any of your desired cell types Those reporting lentivirus has a luminescent report or a fluorescent report under the Wnt responsive promoter that contains 8xTcf tandem repeats as the transcriptional response element The Tcf repress the report expression in the absence of the Wnt signal inducer Once the inducer like Wnt3A prot
7. ium containing antibiotic or perform fluorescence cell sorting followed by antibiotic selection Ready to use Lentiviral Particles for Wnt signaling product manual Page 3 of 6 www gentarget com GenTarget Inc copyright 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 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 MOI 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
8. 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 Ready to use Lentiviral Particles for Wnt signaling product manual Page 4 of 6 www gentarget com GenTarget Inc copyright 2015 7930 Arjons Drive Suite B San Diego CA 92126 e arae ic Phone 858 6788683 Fax 800 3804198 Email orders gentarget com 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 Safety Precaution Gentarget lentiviral particles adapts must advanc
9. permeant CRE recombinase recombinase with different flurescent and antibiotic markers Premade lentivirus expressing LoxP GFP Stop LoxP RFP cassette used to monitor the CRE recombination event in vivo Preamde lentivirus express humanzied wild type Cas9 endonuclease for genomic editing with CRISPR Premade lentivirus expressin TetR tetracycline regulator protein the repressor protein for the inducible expression system Premde lentivirus for human and mouse iPS Myc NANOG OCT4 SOX2 FLF4 factors with different fluorescent and antibitoic markers 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Fax 800 3804198 Email orders gentarget com cell organelle for living cell imaging LacZ Luciferase and more Premade lentivirus expression human or mouse precursor miRNA And anti miRNA lentivector and virus for human and mouse miRNA Premade negative control lentivirus with different markers serves as the negative control of lentivurs treatment for validation of the specificity of any lentivirus target expression effects Ready to use lentivirus expressing specific enzymes with different selection markers www gentarget com copyright 2015
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