GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. Day 3 At 72hr after transduction check the transduction rate via fluorescence image with a suitable filter under fluorescent microscope or calculate the exact transduction rate via Flow Cytometry System FACS or any flow cytometry such as Guava machine Day 3 optional Transduced cells can be sorted out via FACS selected by its specific antibiotics A pilot experiment should be done to determine Ready to use lentiviral Particles for anti miRNA product manual Page 4 of 7 www gentarget com GenTarget Inc Copyrights 2013 6640 Lusk Blvd Suite A107 San Diego CA 92121 en arde ne Phone 858 6788683 Fax 800 3804198 Orders gentarget com the antibiotic s kill curve for your specific cell line Refer to any literatures about How to generate stable cell lines 2 Suspension cells transduction Protocols 1 2 Grow your cell in your completed suspension culture medium shaking in flask in CO incubator if necessary Measure cell density When cell grow to 3 x 10 cell ml measure cell viability should be gt 90 then diluted cells into 1 x 10 cell ml in completed medium Transduction thaw lentiviral particles at room temperature Simply add premade lentiviral particle into the diluted cells at ratio of 50 to 100ul virus per 0 5 ml of cells Note depending on the cell types you may need to use more or less viruses Grow cells in flask shaking in CO2 incubator At 24 hours after transducti2. 6640 Lusk Blvd Suite A107 San Diego CA 92121 en arde ne Phone 858 6788683 Fax 800 3804198 Orders gentarget com 2 Lewis BP Burge CB Bartel DP 2005 Conserved seed pairing often flanked by adenosines indicates that thousands of human genes are microRNA targets Cell 120 1 15 20 3 Hammond S M Dicing and slicing The core machinery of the RNA interference pathway FEBS Lett 2005 579 5822 5829 4 NIH Guidelines for Biosafety Considerations for Research with Lentiviral Vectors Link arranty 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 Product Product Description Category please click links below to see product pages Fluorescent Premade Lentivirus for GFP CFP YFP RFP protein 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
3. 40 Lusk Blvd Suite A107 San Diego CA 92121 ell orgel ne Phone 858 6788683 Fax 800 3804198 Orders gentarget com 2 pre made anti miRNA lentivirus Gentarget s anti miRNA lentivirus are featured with different promoter H1 or U6 and different selection marker an antibiotic marker or a fluorescent antibiotic fusion dual marker providing tools for long term constitutively or inducibly inhibit targeted miRNA functionality in long term stable expression manner The human H1 and U6 promoter are active in almost all types of mammalian cells And lentivirus can transduce into majority of mammalian cell types Thus anti miRNA lentivirus is the easiest most reliable and consistent delivery method for anti miRNA molecular Gentarget s provides the packaged anti miRNA lentivirus to any specific human mouse or any other species miRNA Click to see GenTarget s anti human miRNA list and anti mouse miRNA list Each anti miRNA encodes a specific antisense RNA transcript A negative control anti miRNA a miRNA Neg control lentivirus encodes a deisgned control sequence that has minimal non specific effects which serves as the negative controls for lentivirus treatment All lentiviruses demonstrate the strong transduction efficiency Each virus was validated in lot by lot basis with virus titer at around 1x107 IFU ml and its quality is guaranteed The pre made lentivirus are provided in DMEM medium with 10 FBS and 60ug ml polybrene as 0
4. 5ml rach aliquots in ready to use status or upon request in PBS solution as in vivo ready status For general questions about our ready to use lentiviral particles please see FAQ for pre made lentiviral particles pdf on our website http www gentarget com pdf FAQ Premade Lentiviral particles pdf Note GenTarget also provides pre miRNA expression lentivirus for any specific miRNA expression please contact us for a service quote 3 Why use GenTarget s anti miRNA lentivirus 1 Best delivery method and stable anti miRNA expression lentivirus can be effectively transduced into most dividing and non dividing cells and the anti miRNA can integrate into the host cell s genome for stable long term expression 2 The human H1 and human U6 promoter are active in almost all types of mammalian cells The lentivirus can be used as constitutive high shRNA Ready to use lentiviral Particles for anti miRNA product manual Page 3 of 7 www gentarget com GenTarget Inc Copyrights 2013 6640 Lusk Blvd Suite A107 San Diego CA 92121 en arde ne Phone 858 6788683 Fax 800 3804198 Orders gentarget com expression or optionally used as tetracycline inducible shRNA expression for H1 promoter only Full coverage you can order any anti miRNAs that listed in miRBase from any species simply provide us the miRNA ID or the mature sequence or your own validated anti miRNA sequences Flexible options with the antibiotic m
5. 6640 Lusk Blvd Suite A107 San Diego CA 92121 Phone 858 6788683 GenTaruet inc Fax 800 3804198 Orders gentarget com Pre made anti microRNA for human and mouse lentivirus User Manual Catalog Product Name Amounts A miRNA H1 Bsd anti miRNA H1 with Blasticidin marker A miRNA H1 GB anti miRNA H1 with GFP Blasticidin marker A miRNA H1 GP anti miRNA H1 with GFP Puromycin marker 0 5 ml of anti A miRNA H1 Puro anti miRNA H1 with Puromycin marker SPS ang A miRNA H1 RB_ anti miRNA H1 with RFP Blasticidin marker a cine A miRNA H1 RP anti miRNA H1 with RFP Puromycin marker A miRNA U6 Bsd Anti miRNA U6 with Blasticidin marker 0 5 0 5ml of Negative A miRNA U6 GB Anti miRNA U6 with GFP Blasticidin marker A miRNA U6 GP Anti miRNA U6 with GFP Puromycin marker A miRNA U6 Puro Anti miRNA U6 with Puromycin marker A miRNA U6 RB Anti miRNA U6 with RFP Blasticidin marker A miRNA U6 RP Anti miRNA U6 with RFP Puromycin marker control anti microRNA lentivirus Storage lt 70 C avoid repeat freeze thaw cycles Stable for gt 6 months Product Description 1 Introduction Lentiviral system is a gene delivery tool using lentivectors for gene expression or knockdown Lentivectors are HIV 1 Human Immunodeficiency Virus 1 derived plasmids used to generate lentiviral particles lentivirus that
6. LoxP Premade lentivirus expressing LoxP GFP Stop LoxP RFP ColorSwitch cassette used to monitor the CRE recombination event in vivo 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 Human and Premade lentivirus expressin hundred of human and mouse mouse ORFs ORFs with RFP Blastididin fusion dual markers Living cell Pre made lentivirus particles for Cell Organelle imaging for imgin Nucleus Cytoplasm Endoplasmic Reticulum Golgi Mitochondria Nuclear membrane Peroxisome Plasma membrane Microtubule Chromatin Annexin Actin Connexin and more Fluorescent Pre made lentivirus expression a GFP RFP CFP ORF fusion ORF fusion target shRNA Premade shRNA lentivirus for knockdown a specific genes P53 lentivirus LacZ Luciferase and more microRNA Premade lentivirus expression human or mouse precursor Ready to use lentiviral Particles for anti miRNA product manual Page 6 of 7 www gentarget com GenTarget Inc Copyrights 2013 6640 Lusk Blvd Suite A107 San Diego CA 92121 el arde ne Phone 858 6788683 Fax 800 3804198 Orders gentarget com and anti miRNA And anti miRNA lentivector and virus for human and micr
7. arkers selected from Puromycin blasticidin or dual markers as GFP puromycin GFP blasticidin RFP puromycin RFP blasticidin Transduction Protocols 1 Adhesive cells Transduction Protocols Note the pre made lentivirus is provided as ready to use Simply add certain amount of virus into your cell culture The amount of virus to add is depends on the cell types A quick transduction protocol is add 50ul virus into one well in 24 well plate where cell density is at 50 75 At 72 hours after virus added no need to change medium visualize the positive rate under fluorescent microscope For stable cell line generation pass cell into antibiotic containing medium or sort the cells via fluorescent signal Then select the cells by antibiotics Day 0 Seed the desired cells in complete medium at appropriate density incubate overnight Note at the time of transduction it grows to 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 Add fresh warmed complete medium 0 5ml Thaw the Pre made lentiviral stock at room temperature Add appropriate amount of virus stock to obtain the desired MOI Return cells to 37 C CO incubator Try to avoid thaw and freeze cycles for pre made lentivirus But if you cannot use all virus in one time you still can re freeze the virus at 800C for future use But virus titer will decrease by 10 for each re thaw
8. can be transduced into virtually all kinds of mammalian 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 cells genome for long term expression Therefore lentivirus holds unique promise as gene transfer agents The microRNA miRNA is a small non coding RNA molecule found in plants and animals They are transcripted from RNA precursors pre miRNA and matured at around 19 26 nucleotides in length There are more than 2000 mature miRNA discovered in human and more than 1200 miRNA in mouse so far miRNA mainly silence or repression gene expression via binding to the complementary sequences within gene coding mRNA Each miRNA can target multiple genes It is believed than gt 60 mammalian gene Ready to use lentiviral Particles for anti miRNA product manual Page 1 of 7 www gentarget com GenTarget Inc Copyrights 2013 6640 Lusk Blvd Suite A107 San Diego CA 92121 ell arugi ne Phone 858 6788683 Fax 800 3804198 Orders gentarget com expression was regulated by miRNAs which involves in most biological processes including a variety of diseases or disorders development Anti miRNAi or miRNA inhibitor are short RNA molecules complementary to the mature miRNA sequence Anti miRNA specifically binds to endogenous miRNA which inhibitor or down regulate miRNA activity Therefore anti miRNA is great tool for th
9. e lost of function research of any specific miRNAs It has potential as therapeutic agents for some biological malfunctions Anti miRNA can be delivered by two methods 1 use single stranded nucleic acids designed to specifically bind to the target miRNA sequence or 2 use vector based expression to deliver the antisense specifically bind to the target miRNA sequence GenTarget s ready to use anti miRNA lentiviurs are produced from its optimally designed anti miRNA _ lentivectors The annealed DNA oligonucleotides hairpin duplex encoding the antisense of a specific miRNA sequence was cloned under a constitutive human U6 promoter or under an optional inducible human H1 promoter GenTarget s anti miRNA lentivector was carefully designed for highest anti miRNA expression level with precise promoter transcription position and favor the antisense strand for RISC complex processing The designed asymmetric duplex transcribes a perfect matched antisense RNA molecular to miRNA and leftover a buldged sense RNA molecular The antisense RNA molecular is expressed in high levels and complementarily binds to the endogenous matured miRNA which inactivates targeted miRNAs Please see map scheme below for the anti miRNA lentivectr s core structure anti miRNA lentivector core structure Anti miRNA duplex Ready to use lentiviral Particles for anti miRNA product manual Page 2 of 7 www gentarget com GenTarget Inc Copyrights 2013 66
10. oRNA mouse miRNA lentivirus Negative Premade negative control lentivirus with different markers controls serves as the negative control of lentivurs treatment for validation of the specificity of any lentivirus target expression effects Ready to use lentiviral Particles for anti miRNA product manual Page 7 of 7 www gentarget com GenTarget Inc Copyrights 2013
11. on add equal amount of fresh medium containing related antibiotics Note each particles contain an antibiotic marker and the antibiotic amounts to use depends upon cell types Grow cell in CO incubator At 72 hours after transduction check fluorescence under microscope or calculate the transduction efficiency using cell sorting machine like FACS or Guava machine You can sort the fluorescent positive cells and maintain the antibiotic selection to generate stable cell lines Note Filter wavelength settings GFP filter Ex450 490 Em510 525 RFP filter Ex545 Em620 CFP filter Ex436 Em48s0 YFP filter Ex500 Em535 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 I 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 Lee RC Feinbaum RL Ambros V December 1993 The C elegans heterochronic gene lin 4 encodes small RNAs with antisense complementarity to lin 14 Cell 75 5 843 54 Ready to use lentiviral Particles for anti miRNA product manual Page 5 of 7 www gentarget com GenTarget Inc Copyrights 2013
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