User Manual PDF - ATCGbio Life Technology
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1. 5 The kits provide enough reagents for 3 times AdenoComplete ADIOOI amp AD1002 and 9 times AdenoEssential AD1003 purification 6 In AdenoComplete AD1002 we provide crude Adeno GFP expression vector as a positive contro 7 In vivo compatibility The injection of adenoviruses purified by our kits to mouse through the tail vein shows the efficient transduction in the liver without apparent immunoreactions 8 Very economical We set up the most cost effective way to purify adenovirus without compromising performances 9 Detailed protocol describes the safe preparation and usage tp wwwategbio com Page 2 Adeno Purification Kits AD1001 AD1002 amp AD1003 Products and Related Products ATCGbi LIFE TECHNOLOGY IN ADIOOI AdenoComplete Purification Kit preps ADIOOZ AdenoComplete Purification Kit with Adeno GFF 3x preps ADIOOS AdenoEssential Purification Kit 3 x preps ADIDI AdenoRetriever Solution SOOT vial ADIOI2 AdenoBooster Solution S00 ivial ar teria OT S00 vial ADI020 Crude Adeno GFP Expression Vector Loe 10 pit Grade Adeno B Galactosidase Expression Vector S00 Tvial eee 1 0 x 10 pfvent Kits Components Catalog AdenoComplete ADI001 GAD1002 AdenoFssential ADI003 ADIOO1 OF Blunt needle 3 Blunt needle r9 ADIOO 02 Binding fer T Binding filter x 3 AD1001 05 30ml Loading Buffer 7 T 100ml Loading Buffer lt2. Contact Information Laboratory 3938 North Fraser Way Burnaby BC V5 SH6 Canada Tel 01 778 321 9336 order only Fax 01 617 566 1092 order only Email info atcgbio com for order and sales tech ategbio com for technique support Business Hours Monday to Friday 3am 5pm GMT 8 00 Pacific US Ordering information All of your orders are available on line at http www ategbio com Technical Support Please send email to us tech ategblo com We will response within 1 2 business days hitpi www ATCGbio com Page 11
3. T ADIOOI 0477 60ml Washing Buffer T 5 concentrated 60ml Washing Buffer T AD1001 04 5 ADIOOI0S TSial Elution Buffer T 0 ml Elution Bulfor x T ADIO01 06 1 Sml Stabilizer Solution T keep ar FC _ 1 Sml Stabilizer Solution x 2 keep a C ADIO 10 Sml Booster Solution T keep at C 1 5 ml Booster Solution gt T keep at 4 C For product AD T002 the kit supplied with Crude Adeno GFP expression vector in 2ml tube and shipped at room temperature store at 20 C once received Catalog number AD1001 04 is for 1x Washing Buffer Catalog concentrated Washing Buffer Materials and Stericup Gi Equipments Users Provide number ADIOOI 04 5 is for 5x Y 0 22pm 150ml catSCGVUOIRE from Millipore S7 00 each Disposable Luer Lock syringe 20ml 10ml and 3ml such as BD 309661 BD 309604 and BD 309585 For safety reason Luet Lock syringe should be used 7 5 NaHCO3 such as Invitrogen 25080 094 may need to adjust pH 50m sterile tube to collect crude adenovirus 15m sterile tube to collect purified adenovirus Centrifuge machine 2000 rpm accommodating 50 ml tube Water bath 30 C or 37 Bleach 10 Bleach solution diluted concentrated bleach with water 500m beaker to trash used materials and treat with bleach inside Decant bottle The bottle is for collect medium after virus binding 50 ml tube also can be used instead of the bottle Biosafety cabinet Class I
4. pfu Calculate OD20 Dilution Factor x 2 x 10 pfu ml For example if OD 0 100 the titer is 0 100 gt 20 x 2 10 4 0 x 10 pfw mt hrtpiwww ATCGbio com Page B Adeno Purification Kits AD1001 AD1002 amp AD1003 ATOGbio LIFE TECHNOLOGY INC Terminology for Adenovirus Work Pfu or Ifa The plaque forming unit pfu is the number of viral particles capability to form the plagues the area of cll Iysis by per viral particle It isa functional assessment rather than the absolute quantification of viral particle numbers viral particles that are defective or which fail to infect their target cells will not produce a plaque and thus it will not be counted For example a solution of adenovirus with the concentration of 1 000 pfu yl means that there are 1 000 infectious viral particles in one microliter of the solution In case of adenovirus the procedure for quantified pfu takes 3 weeks in HEK 293 cells Iu infectious unit can also be used instead of pfu Ideally both give the same number Current popular method to estimate this number is to count the HEK 293 cells number expressing polyhedron protein adenovirus envelop after infection Estimated Pfu or Ifu from ODs Using the virus prepared by a traditional centrifugation method Challberg and Ketner reported that pfi can be estimated directly ftom OD by the formula pl 00260 dilution factor 3 5 x 10 However adenovirus prepared by our kit has muc
5. infecting the cells 1 Freeze and thaw 2 centrifuge 3 Filter 4 Add the lacie onioe dyke loading buffer e e e e j S Precondition 6 Bind 7 wash s elute preniter Binding fiter L 4 3 tpl wwwategbio com Page 5 Adeno Purification Kits AD1001 AD1002 amp AD1003 ATCGbi LIFE TECHNOLOGY INC Safety Precautions Use personnel protection equipment Lab coat gloves and safety glasses Always open the adenovirus container inside a safety cabinet Kill adenovirus with concentrated or 10 bleach Do work in the biosafety level 2 facility regulated by the NIH Office of Biosafety and Canadian Laboratory Biosafety Guideline 5 Follow regulations set by your institute gt Notice for first time user The kit uses Luer lock syringe to withdraw virus solution and buffers into the syringe and apply the solution through the filters If you are not familiar with Luer lock syringe it is better to practice just before purification using 20ml syringe and a blunt needle supplied with kit To attach syringe take syringe by the right hand and twist it clockwise and to detach syringe twist it anti clockwise Attaching blunt needle Remove a gray cap from the needle holder Take a needle with left hand and a syringe with right hand Attaching each other and twist syringe clockwise Pull syringe from needle holder Detaching blunt needle 41 Take a needle holder with left
6. 200 ml Sterile water for AdenoEssential kits Spectrophotometer capable to measure OD260 011 SDS dissolved in Dulbecco s PBS DPBS or TE buffer OLSN NaOH 10ml for regeneration of the binding IN NaOH solution If DMEM with 25mM HEPES is used to culture 293 cells 20 Ethanol in DPBS Add 40ml DPBS to 10m1 100 ethanol ntpa wwwategbio com Page 3 Adeno Purification Kits AD1001 AD1002 amp AD1003 ATCGb LIFE TECHNOLOGY IN Adenovirus Amplification in HEK 293 Cells Getting high titer adenovirus depends on HEK293 cells condition We recommend to culture HEK293 cells with 10 FBS FCS high glucose 4 5 g L DMEM under 5 10 CO at 37 C in 10em dishes or 1 75 flask IFuser use DMEM containing 25mM HEPES IN NaOH would be added during purification procedures describe later Healthy HEK293 cells exhibit flat epithelium morphology as shown below Prepare one seeding plate a plate for splitting Once it grows to 80 90 confluence split it to 5 of 10cm dishes equally with each 10ml medium one for seeding and others for amplification Typically it is not necessary to change medium until the cells reach 80 90 confluence Ater the cells reach about 90 confluence i s ready for amplification Place 10 mi fresh medium and infect the cells with 5 10 MOI of adenovirus If you don t know the viral titer you should first determine the virus amount that cause the cytopathic effect CPE in one well of 6 wel
7. A TLGb0 Welcome to the Next Level of Adenovirus Pu User manual AD1001 AdenoComplete Purification Kit 3 preps AD1002 AdenoComplete Purification Kit 3 preps with Adeno GFP AD1003 AdenoEssential Purification Kit 9 preps Version 1 1 ATCGbio LIFE TECHNOLOGY INC Adeno Purification its AD1001 AD1002 amp AD1003 ATCGbie LIFE TECHNOLOGY INC Content Introduction Products and Related Products Kits Components Materials and Equipments Users Provid Adenovirus Amplification in HEK 293 Cells Steps at a glance Safety Precautions gt Notice for first time user Detailed Protocol Regeneration of the Binding Filter Viral Titration Terminology for Adenovirus Work gt Examples for viral titration by using our kits Usage of AdenoBooster Solution Table1 Approximate Cells Yield References Contact Information FOR RESEARCH USE ONLY Not for use in clinieal or diagnosis purpose Impor ce Even the recombinant adenovitusos are replication deficiant by deletior adenovirus has baen classified in biosafety laval I for agents considered of ordinary potential harm and you need BL 2 vel facity to work wih according to references issued by the NIM Office of Biosafety It shouid be noted that coll culture facies in most insttutes ar certified as BL 2 leval Wid type replication competent adenoviruses could cause cold symptoms but
8. also could be used for in vivo injection to mice Usage Add 1 10 AdenoBooster Solution to adenovirus solution prepared by our kit Note just before use For example adding 1l AdenoBooster solution to 10 yl of virus solution and incubate at RT for 5 10 min just before infection Ithe virus amount is less than 10 pl dilute 10 times of AdenoBooster Solution with sterile DPBS just before infection and add 1 1 ratio of AdenoBooster solution virus solution For your convenience we also offer reporter crude adenoviruses expression vectors such as 8 Galactosidase AD1030 or GFP AD1020 to evaluate infection efficiency in your target cells Table1 Approximate Cells Yield This table provides approximate cell number in various plates and flasks If you don t know the cell number to be infected use this table Plate Number ofthe cells well Plate Flask Number of the cells 24 well S010 60mm 32x10 12well Loto 100mm iaa well 25x10 T5 20107 35mm 2 0 10 TIs0 6 0107 hitpi mww ATCGbio com Page 10 Adeno Purification Kits AD1001 AD1002 amp AD1003 ATCGbio LIFE TECHNOLOGY INC References Cacicedo JM Yagihashi N Keaney JF Jr Ruderman NB Ido Y Biochem Biophys Res Commun 2004 Nov 26 324 4 1204 9 Lan F Cocicedo JM Ruderman N Ido Y 4 Biol Chem 2008 Oct 10 283 41 27628 35 Cacicedo JM Benjachareonwong Chou E Yagihashi N Ruderman NB Ido Y Invest Ophthalmol Vis Sci 2011 Jun 1 52 6 3630 9
9. bind to the binding filter Speed is not so critical Repeat until all the adenovirus solution passed through Trash the 10 ml syringe and the needle into a beaker 13 Withdraw remaining 17 ml washing buffer into a 20 ml syringe Push the washing buffer through the binding filter at a rate of 1 3 drops per a second Once again speed is not critical Fig 6 next page sae ttp www ategbio com Page 7 Adeno Purification Kits AD1001 AD1002 amp AD1003 ATCGbio LIFE TECHNOLOGY INC 14 To elute the purified adenovirus from the binding filter use a Figs 15ml tube sterilized and place the binding filter on the top of the tube Take 2ml of the elution buffer into a 3ml syringe Push the elution buffer through the binding filter at a rate of 1 2 drops per second Need to be slow Fig 7 Stop after mil buffer is through and wait for min then finish the rest of the solution Take air into the syringe and push the air through the filter to complete the elution 15 Add 1 10th of the Stabilizer solution 0 2 0 25 ml and aliquot it typically 300 pl each into the sterile vials you like and keep them at 80 C until use 16 Estimate virus titer and regenerate the Binding Filter 17 Finishing up Add concentrated bleach into a decant bottle to kill any remaining viruses by waiting for 10 min Treat used materials in the beaker with 10 bleach Dispose all the materials into biosafety bin a
10. cal changes that the cells undergo after infection Infected cells typically remain intact but round up and may detach from the dish individually or in grape ike clusters that float in the medium tpl wwwategbio com Page 4 Adeno Purification Kits AD1001 AD1002 amp AD1003 ATCGbi LIFE TECHNOLOGY IN Steps at a glance ihe detailed protocol at page 9 Thaw and freeze thaw two times thaw and one time freeze Centrifuge 5 min at 2000 rpm 500 1000 g at room temperature to precipitate debris Filtrate adenovirus containing medium through a filtration unit Add the Loading Buffer with 1 10 volume of filtered medium contain adenovirus e g add 4ml if the medium volume becomes 40ml after filtration 5 Precondition the binding filter with 3 4ml 1x washing buffer for AdenoEssential Kit users first dilute x5 washing buffer 4 ml with 16 ml sterile water in 50 ml conical tube 6 Filtrate medium containing adenovirus through the binding filter using 20ml Luer lock syringe Adenovirus will be bound to the binding filter 7 Wash the binding filter with ISml Lx washing buffer 5 Elute purified adenovirus by applying 2ml elution buffer 9 Add the Stabilizer Solution with 1 10 amount of purified adenovirus about 2001 10 Measure the absorbance at 260nm to estimate virus titer 11 Aliquot out adenovirus to proper vials and store them at 80 C 12 Ifnecessary use AdenoBooster Solution cat AD1012 just before
11. e 5 min at room temperature j Bring all the materials inside a biosafety cabinet Fig 2 Connect the vacuum with the vacuum filter unit Stericup GV 0 224 150 ml Estimate volume of the adenovirus solution you plan to purify will be 40 90m Apply adenovirus solution to the center of the filter 8 Use a Sml sterile pipette add the Loading Buffer into the filtered adenovirus solution The amount of Loading Buffer you add is 1 10 volume of the estimated adenovirus solution in step 7 e g if the estimated adenovirus solution is 40ml add 4ml loading buffer The solution color should tum red to purple If the DMEM with 25mM HEPES is used in HEK293 cell culture add also 1 100 of IN NaOH to bring up pH e g 0 4 ml of IN NaOH 10 40 mi filtered solution 9 Condition the Binding Filter In case of AdenoComplete user wansfer 20 ml Washing Buffer into SOml tube sterilized In case of AdenoEssential user dilute 4 ml of S Washing Buffer With 16 ml sterile water Take 2 3 ml Washing Buffer directly into 10ml syringe Don t need to use a needle and attach the Binding Filter Push the solution through the filter into a decant bottle Fig 10 Detach the syringe from the filter and attach a blunt needle 1 Withdraw the adenovirus solution into the 10 ml syringe Fig4 12 Detach the blunt needle attach the Banding Filter and push the adenovirus solution through the filter Fig S at a rate about 1 3 drops per second virus will
12. generally da not cause serious iiness Far more Information on biosafety levels please visit hilipsioba od nin gow in the E1 and E3 regions recombinant human tps wwwategbio com Page 1 Adeno Purification Ki AD1001 AD1002 amp AD1003 ATCGbie LIFE TECHNOLOGY IN Introduction Thank you for purchasing ATCGbio products Adeno Purification Kits are achievements of our scientists 10 years experience and more than 5 years researches We proudly present and share our kits with you For a new user the kits could be used as the training kits to help the researchers understanding procedure of adenovirus amplification and purification The kits have following features standing inthe top of the adenovirus purification methods currently available 1 Obtain High quality adenovirus in 1 5 hour based on membrane filtration The procedure does not require ulircenttifpation 2 Typically it only requires HEK293 cell culture in 4 of 10 em dishes or T75 flasks can be scaled up to 9 dishes or flasks to yield adenovirus up to 1 0 10 pfu in 2 ml volume 3 Supplied Stabilizer provides an unprecedented stability by virtually eliminating freeze thaw degradation problem up to 10 times 4 upplied AdenoBooster Solution ADI could increase the infection efficiency up to 50 times more for the cells lacking of coxsackie adenovirus receptor CAR or the cells having difficult infection characters
13. h higher quality than those and the formula determined empirically is close to QDs dilution factor x2 x 10 pfwiml MOI Multiplicity of Infection I is the ratio of number of virus to the number ofthe cell An MOI of 10 indicates that there are 10 transduction units Functional virus pfu or ifi for every cell in the well It is important to note that different cell types require different MOTs for successful infection In addition because actual transduction infection occurs through statistical processes even in completely permissive cells like HEK 293 cells MOI 1 produces only 63 infection and MOIS is required for 99 transduction gt Examples for viral titration by using our kits Recombinant adenovirus is a choice to express proteins sKRNAS and microRNAs Since promoter activity could be variable depending on cell types and expressed products RNA and protein may be unstable expression levels are not only determined by the adenovirus titer Therefore it is difficult to describe a protocol work in every situation Followings are guidelines when you want to expresse proteins in cultured Tuman or rodent cells Calculation is based on our empirical data Example 1 We have purified adenovirus by our kit and measured OD 0 100 Using a formula described previous section pfu ml ODsso x dilution factor x 2 10 Estimated titer is 0 1 x 20 x 2x10 4 x 10 pfu ml 4x 10 pfu pl Human endothelial cells are growing co
14. hand and a syringe with right hand 2 Put need holder onto needle 3 Twist syringe anti clockwise FOR RESEARCH USE ONLY Not for use in clinical or diagnosis purpose important Not Even the recombinant adenoviruses ar replication deficiant by deletions in the E1 and E3 regions recombinant human adenovirus has baen classified in biosafety leval Il for agents considered of ordinary potential harm and you need BL 2 Javel faciity to work with it according ta referencas issued by the NIH Office of Biosafety It should be nated that cell culture facies in most institutes are certified as BL 2 leval Wild type replication competent adenoviruses could cause cold symptoms but generally da not cause serious iness Far mare Information on biosafety levels please visit hlipsioba od nih gov tpi wwwategbio com Page 6 Adeno Purification Kits AD1001 AD1002 amp AD1003 ATCGBIO LIFE TECHNOLOGY INC Detailed Protocol Before processing purification please read carefully the following protocol first 1 Take out the adenovirus tube from 80 C or 20 C Make sure te lid is capped tightly 2 Thaw in 30 C or 37 C water bath 1 37 C water bath is used take out immediately before the ice is completely melted Fig Notice Do not eave in 37 C for a long time Tighten the lid again Do not vortex Freeze the tube by putting at 80 C for 20 min Thaw again in water bath like step 2 Fig 1 Centrifige at 2000 rpm fo
15. l plate 90 conference Appropriate amount of the virus should cause the CPE in 3 5 days Then use 6 times more virus for one 10cm dish For example if 1 of your virus can cause your HEK293 cells appearing CPE in one well of 6well plate you need 60pl of your virus for each 10em dish Notice Do not use too much high titer adenovirus to infect the cells e g all the HEK293 cells float just in 1 or 2 days afier infection Once you add adenovirus you should not change medium Very important During course of amplification ifthe medium color becomes yellow indication of lower pH add 50 100 pl of 7 5 NaHCO solution such as Invitrogen 25080 094 to correct pH until the medium color turns to just light purple If there is no sign of the CPE in your HEK 293 cells after 5 days add Lor 2 ml fresh medium and keep incubating When the most of HEK 293 cells float about 90 collect all medium and cells together about 40 50 ml into 50ml sterile plastic centrifuge tube Such as Coming 4558 or Falcon 352070 Keep the tube at 80 C or 20 C and keep it in standing position to avoid accidental leak during freezing until it s ready to be purified by the kit It is possible to keep the solution for a month or more without titer losing Once it is ready please follow the adenovirus purification protocol described in page 7 Gh cells ound wp and cell edge lnk shiny The cytopathic effect CPE refers to the morphologi
16. nd throw bleach treated solution into a sink Wipe with 10 bleach inside a cabinet wait for 10 min and clean the cabinet Regeneration of the Binding Filter The binding filter can be used three times without losing its performance 1 Use a 3 ml syringe to take 3 ml the elution buffer and push through the binding filter 2 Change syringe and take 3 mil 0SN NaOH and push through 1 5 ml and wait for 5 mi the remains through the filter 3 Change syringe and take 3 ml sterile DPBS and push through the filter 4 Take 3 ml 20 ethanol in sterile DPBS and push through the filter Eliminate residual solution by pushing air Put the filter into a clean plastic bag and seal it and keep it at room temperature until use it again then push Viral Titration The virus titer prepared by our kit can be estimated with a spectrophotometer or a Nanodrop by measuring Odes Dilute purified adenovirus solution 20 times with 0 1 SDS sodium dodecyl sulfate dissolved in DPBS or TE to the volume your spectrophotometer can read For example if the machine can measure a volume of 100l take Spl virus solution and dilute with 95 0 1 SDS solution and use Sp elution solution 95y1 SDS as a blank control Measure them at absorbance 260 nm By using SDS virus became inactive and it can be disposed without further treatment Calculate ODasi OD virus OD blank We typically get the number between 0 050 0 200 Estimated virus titer
17. nfluent in a 12 well plate We want infect almost all the cells with moderate expression levels First estimate the number of the cells in one well of 12 well plate Number of cells growing in a well of 12 well plate is about 10 cells See the table1 in page 14 Although this may not be exact it is close enough Second calculate MOI for the cells you use Required MOI for your cells usually differs from HEK 293 cells For HEK 293 cells MOI 5 should infect most of the cells at least with one virus It is good estimate to use MOI 25 for human cells and MOI 125 with AdenoBooster Solution for the cells from other species Third calculate amount of adenovirus per well According to the cell number in one well and MOI it should be 25 MOI x 1 x 10 cell number well 2 5 10 pfu the number of functional virus wel Since virus titer is 4 x 10 pfu ul 2 5 10 pfwwelly 4 x 10 pfu 0 625 pl virus solution well is hitpi mww ATCGbio com Page 9 Adeno Purification Kits AD1001 AD1002 amp AD1003 ATCGbio LIFE TECHNOLOGY INC needed Because the actual protein expression and titer may vary test to 2 folds range of adenovirus It will be 03 0 6 1 2 ulof the virus each well with 1 ml fresh normal culture medium Ifthe adding viral amount is too small to pipette dilute 10 times with DPBS Incubate the cells with virus for overnight and change the fresh medium next day Check protein expression after 48 72 hour
18. s by western blot and or by immunochemistry and determine desired adenovirus amounts Since repeated freeze thaw cycle has virtually no effects on Virus titer prepared by our kit you can use this number for later experiments as far as you keep 80 C after ench use Example 2 The same virus is used to infect mouse C2C12 cells cultured in the 6 well plate Estimated the cell number is approximately 2 5 x10 per well see table I MOI should be 125 and infecting with AdenoBooster Solution So 125 x 2 5 x 10 pfuwell 312 5 x 10 31 x 10 pfuiwel Since virus titer is4 10 pfuul amount of virus is 31 4 7 8 uliwell One well requires approximately 8 pl virus 0 8 pl AdenoBooster How to write in your paper You can report either in MOI or in Pfu In case of Example 1 25 MOI or 5 10 per well or ml or 50 fuel were infected Usage of AdenoBooster Solution Not every type of the cell requires the AdenoRooster Solution ADIO12 The cells lacking of coxsackie adenovirus receptor CAR are refractory for adenovirus infection For these cells we provide the AdenoBooste Solution that is able to enhance the infection efficiency by 10 80 folds The solution has no effects on permissive cells like HEK 293 cells though without any toxicity In our tests human cells usually do not require the solution but most of rodent cells except adult cardiomyocytes and bovine cells require this solution The solution mixture with adenovirus
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