User Manual PDF - ATCGbio Life Technology
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1. Catalog Components Size Shipping and storage SH4001 01 Custom made shRNA Lenti 9 0mlx2 Shipped at room temperature store at Particle Solution 4 C then 20 C or below after aliquot SH3002 shRNA Lentivirus Particle 9 0mlx1 Shipped at room temperature store at Control Solution 4 C then 20 C or below after aliquot SH3003 Storage Solution 1 0ml x1 Shipped at room temperature Keep at Reconstitution Solution 4 C www atcgbio com 2 Recombinant shRNA Lenti Particle Solution SH3001 amp SH4001 ATCGbio Life Technology Inc ka Usage Please make plan first then aliquot out to store To make plan for cells infection follow the guideline below e Keep at 4 C before aliquot Aliquot the lentiviral solution and store at 20 C or below e Storage at 4 C for more than 3 days is not recommended Case 1 Cells Growing in DMEM Compatible Medium Many cells grow in DMEM compatible medium such as the most of cancer cells transformed cells endothelial cells and so on In this case lentiviral solution can be added directly to infect the cells without purification precipitation It is recommended that the cell density should be about 30 at the time of infection 1 Add lentivirus solution directly into the plate where cells are growing 2 Incubate for 16hrs 3 Polybrene is not necessary 4 Follow the suggested amount of lentiviral solution table 1 to achieve near 100 transduction Table 12. User Manual e Pre made shRNA Lenti particle solution SH3001 e Custom made shRNA Lenti particle solution SH4001 ATCGbio Life Technology Inc www atcgbio com Os A es NS Recombinant shRNA Lenti Particle Solution SH3001 amp SH4001 ATCGbio Life Technology Inc gt Contents Product Features aici cisiccsccges tices scnvadisenciads dais vendassdaachacds ica dep iecatacavasendnncanserccaxadsobiedansicashiaersstes 2 Product Components Shipping and Storage Conditions ceeee 2 MS ROS ri aoa oe cate cacti xnewtnecs coms ages cence contesaccnteanatenatte dente cnsnbersencnsnes geet Meesdhan duandunndesmesssnessahenensensescaseieanes 3 Case 1 Cells Growing in DMEM Compatible Medium e 3 Case 2 Cells Growing in DMEM Incompatible Medium 05 3 Hygromycin Selection csccc oe ee tst rntttAtttEnEtErntEEerEnen Eset Ee seent 4 Referent OS 65 iors eee ie i i ee ee 4 Contact Information 000 0 cc cccceecessseeeeeseeaeeeesseeaeeesseceeeeesceseeeessseaeeeeesenaeeeeessaanees 4 FOR RESEARCH USE ONLY Not for use in clinical or diagnosis purpose Important Notice Laboratory workers handling pathogenic lentiviruses recombinant lentiviral vectors naturally or experimentally infected laboratory animals or clinical specimens potentially infected with lentiviruses Diagnostic specimens that contain human blood body fluids or tissues can be handled and manipulated at
3. Size of Culture Culture Medium Lentiviral Solution 1 well of 6 well plate 2 ml 0 5 1 ml 1 well of 12 well plate 1ml 0 25 0 5ml 1 well of 24 well plate 0 5 ml 0 125 0 25 ml 5 To achieve modest infection less than 1 virus per cell reduce viral amount to 1 3 1 5 suggested in table 1 6 After 16hrs incubation change to fresh media to resume normal culture Expression of GFP can be observed in 48 96 hrs after starting infection depending on cell types Knock down effects can be observed in a similar time frame but are also affected by target protein turnover Case 2 Cells Growing in DMEM Incompatible Medium Some cells such as keratinocytes grow in DMEM incompatible medium It is recommended that the lentiviral particle solution should be precipitated purified for those cells infection The cell density should be about 30 at the time of infection Steps for virus precipitation purification and infection 1 Take the amount of viral solution described in table 1 into an appropriate tube micro centrifuge or 15ml tube For example to infect one well of 6 well plate take 1ml 2 Precipitate the lentivirus by centrifuge at 1 500xg for 30 min at 4 C The pellet may be or may not be visible 3 Take a pipette out of the supernatant carefully www atcgbio com 3 Recombinant shRNA Lenti Particle Solution SH3001 amp SH4001 4 Decant residual supernatant by upside down the tube for 2 3 minutes and re
4. the BSL 2 level BSL 2 is also appropriate for generating and using lentiviral vectors and handling animals and animal tissues blood body fluids and cell lines that are infected with lentivirus vectors When you practice recombinant lentiviral vectors please following the requirements outlined in http oba od nih gov rdna_rac rac guidance _lentivirus html and CDC Biosafety in Microbiological and Biomedical Laboratories 5th edition the NIH Guidelines for Research Involving Recombinant DNA Molecules latest edition www atcgbio com 1 ae lt S 4 T Recombinant shRNA Lenti Particle Solution SH3001 amp SH4001 ATCGbio Life Technology Inc lt y7 The User Manual Pre made shRNA Lenti particle solution SH3001 Custom made shRNA Lenti particle solution SH4001 Product Features shRNA Lenti particle solutions SH3001 amp SH4001 are recombinant shRNA expression lentivirus ready to infect directly It s specifically designed for scientists who don t have time or don t prefer to make lentivirus by themselves It s the easiest way to knockdown gene of interest shRNA Lenti particle from ATCGbio Life Technology are created base on safer ch generation recombinant lentivirus vector and our new shRNA design system and produced by our own Lentivirus Production Kit LT1001 Virus expresses shRNA for target gene GFP protein and hygromycin resistance gene product It is suitable for both cultured cells and in vivo in
5. jection to small animals Lentivirus solution is DMEM based medium containing 2 FBS and our lentivirus precipitation purification solution It does not contain any chemicals used to boost lentivirus titers such as chloroquin acetate or nicotines The solution can be used directly to the cells growing in DMEM compatible medium without further precipitation purification There is no visible viral toxicities e g change in cell morphology or and cell detachment from culture plate if the viral solution added up to one third of total culture medium as described in table 1 Some special cells such as keratinocytes may be needed to grow with special medium which is incompatible with DMEM or FBS In such a case it is recommended to precipitate purify lentivirus and reconstitute it with storage solution as described below Product Components Shipping and Storage Conditions Pre made shRNA Lenti particle solution SH3001 350 00 Catalog Components Size Shipping and storage SH3001 01 Pre made shRNA Lenti Particle 9 0 mlx 1 Shipped at room temperature store at Solution 4 C then 20 C or below after aliquot SH3002 shRNA Lentivirus Particle 9 0mlx1 Shipped at room temperature store at control solution 4 C then 20 C or below after aliquot SH3003 Storage Solution 1 0 ml x1 Shipped at room temperature Keep at Reconstitution Solution 4 C Custom made shRNA Lenti particle solution SH4001 350 00
6. ly Fax 01 617 566 1092 order only Email info atcgbio com Business Hours www atcgbio com 4 b A A _ lt gt Recombinant shRNA Lenti Particle Solution SH3001 amp SH4001 ATCGbio Life Technology Inc Q Monday to Friday 9am 5pm GMT 8 00 Pacific US Ordering information All of your orders are available on line at https atcgbio com Technical Support Please send email to us info atcgbio com Or click Contact Us to fill the form for enquires We will response within 1 2 business days www atcgbio com 5
7. suspend by adding the amount of storage solution described in table 2 into the bottom of the tube Table 2 Lentivirual Solution Used Storage Solution Needed 1 0ml 50ul 0 5ml 25 ul To infect more wells increase the storage solution proportionally For example to infect cells in all of the 6 wells of the 6 well plate after centrifuge 6ml of viral solution step 2 use 300ul storage solution to re suspend step 4 5 Incubate at 37 C for 10 minutes and briefly mix by pipet tips Add the solution directly to the cultured cells and incubate 16hrs Then change to fresh media to resume normal culture Unused lentivirus re suspended with storage solution may be stored in 80 C Hygromycin Selection Hygromycin selection 50 100 ug ml at final concentration can be initiated after confirming transduction by GFP expression which takes 2 5 days after infection depending on cell type Following procedures can accelerate selection 1 Incubate with hygromycin overnight 2 Split the cells trypsin EDTA appropriate ratio 1 to 2 or 1 to 3 and cultured with the medium containing hygromycin Only the cells expressing resistance gene will re attach on the plate under hygromycin condition References 1 Y Ido et al PLoS ONE 2012 Apr 07 4 e35092 2 LanF etal J Biol Chem 2008 Oct 10 283 41 27628 35 Contact Information Laboratory 3938 North Fraser Way Burnaby BC V5J 5H6 Canada Tel 01 778 321 9336 order on
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