AAVpro® Purification Kit (All Serotypes)
Contents
1. Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da VI 2 Purification and concentration of AAV particles Note Use swinging bucket rotors for steps VI 2 5 VI 2 6 and VI 2 7 if Add 1 100 volume of Cryonase Cold active Nuclease to the solution from step VI 1 10 and then incubate at 37 C for 1 hr Add 1 10 volume of Precipitator A vortex for 10 sec incubate at 37 C for 30 min and vortex again for 10 sec Note 1 Precipitator A may produce a white precipitate at low tempera tures however this does not affect the quality or performance of this reagent If a precipitate is present dissolve it completely at 37 C before use Note 2 Although a precipitate may form during the reaction this is not a problem Proceed to the next step Add 1 20 volume of Precipitator B to the solution promptly vortex for 10 sec and then centrifuge at 5 000 9 000 X g for 5 min at 4 C Note A precipitate is formed after adding Precipitator B proceed to cen trifugation Filter the supernatant using a Millex HV 0 45 um filter Add the filtrate containing AAV vector to an Amicon Ultra 15 100 kDa filter Centrifuge at 2 000 X g for 5 min at 15 C and then confirm that the AAV solution in the filter is lt 1 5 ml Note If the volume of the solution is gt 1 5 ml continue to centrifuge After removing the filtrate add 5 ml of Suspension Buffer inside the cup of the Amicon Ultra 15 filter unit and mix the solution unifor2. 5 Cat 6664 AAVpro Packaging Plasmid AAV6 Cat 6665 AAVpro Titration Kit for Real Time PCR Ver 2 Cat 6233 EDTA Buffer Powder pH8 0 Cat 19191 AAV 2 Cat 6230 AAV5 Cat 6650 AAV6 Cat 6651 AAV2 CRE Recombinase Cat 6652 AAV5 CRE Recombinase Cat 6653 AAV6 CRE Recombinase Cat 6654 AAV2 LacZ Cat 6655 AAV5 LacZ Cat 6656 AAV6 LacZ Cat 6657 AAV2 U6 ZsGreen1 Cat 6658 AAV5 U6 ZsGreen1 Cat 6659 AAV6 U6 ZsGreen1 Cat 6660 NN on oN oN Ne eS ee Note Not all products are available in all regions Please confirm availability in your region AAVpro is a registered trademark of TAKARA BIO INC Cryonase is a trademark of TAKARA BIO INC NOTE This product is for research use only It is not intended for use in therapeutic or diagnostic procedures for humans or animals Also do not use this product as food cosmetic or household item etc Takara products may not be resold or transferred modified for resale or transfer or used to manufacture commercial products without written approval from TAKARA BIO INC If you require licenses for other use please contact us by phone at 81 77 565 6973 or from our website at www takara bio com Your use of this product is also subject to compliance with any applicable licensing requirements described on the product web page It is your responsibility to review understand and adhere to any restrictions imposed by such stateme
3. 700 2 000 X g for 10 min at 4 C and discard the supernatant Centrifuge again at 1 700 2 000 X g for 1 min at 4 C and remove the super natant completely Note Be sure to completely remove the supernatant because remaining supernatant could impair virus purification Loosen the cell pellet well by tapping or vortexing Note If the cell pellet is not loosened sufficiently the purification effi ciency can be decreased Make sure there are no cell clumps before proceeding to the next step Add 10 ml of AAV Extraction Solution A plus Resuspend for 15 sec by vortexing Note Continue to vortex until there are no remaining cell clumps Incubate for 5 min at room temperature and then vortex again for 15 sec Centrifuge at 4 000 9 000 X g for 10 min at 4 C Note In some cases the efficiency can be improved by repeating steps 7 9 Collect supernatant in a new sterile centrifuge tube using a pipet to avoid contamination Add 1 10 volume of AAV Extraction Solution B Note 1 The sample can be stored at 80 C at this stage Alternatively promptly proceed to step VI 2 1 If the sample is stored at 80 C thaw in a 37 C incubator before using Be sure to use a tube that is resistant to freezing and centrifugation when a sample is stored at 80 C Note 2 When AAV Extraction Solution B is added the color of the solution may turn pink in some cases this does not affect performance URL http www takara bio com TAKARA BIO INC
4. arch involving recombinant DNA Take appropriate safety measures when producing or handling recombinant adeno associated viruses including working in a biological safety cabinet and wearing protective laboratory coats face protection and gloves URL http www takara bio com TAKARA BIO INC Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da l Introduction Adeno Associated Virus AAV is a non enveloped virus that belongs to the Parvovirus family of the Dependovirus genus There are more than 100 serotypes of AAV and the host specificity and char acteristics of the virus differ among serotypes AAV is not thought to be pathogenic to humans and only replicates in the presence of a helper virus such as adenovirus or herpesvirus The AAV genome is a linear single strand DNA molecule of approximately 4 7 kb Adeno associated virus vectors AAV vectors exploit the properties of AAV for transduction of genes to cells and organisms AAV vectors are used as research tools and also as vectors for gene therapy In addition AAV vectors are generally considered safer than adenoviral and retroviral vectors AAV vectors can be used to transduce genes into both proliferating and non proliferating cells and can impart long term expression in non dividing cells In addition AAV has little immunogenicity and is suitable for the transduction of genes into animals as an in vivo transduction tool ll Description The purity an
5. ca 6666 For Research Use TahaRa AAVpro Purification Kit All Serotypes Product Manual v201508Da Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da Table of Contents l ERO dUCti OMen DD nnn 4 Il DESCO 0 g siy Pn nin nen nea ton n n K r h G k e nner renee tee 4 llli Go BGS 6 LAR See D rr E 6 V Materials Required but not Provided uuEmmmmmeeeeeeee 6 LIR Ho tela ol Dy DD r r rrr 7 VII Experimental Examples xssccccsssccustesaiapseacvatins sceasaitbolgarseaseuncetecntviacrente 9 VIII Related products ssssssssssesssssssseessssssssssssosossooossssssssonnnnnnnsooreeeesssssssssssnnnne 11 TAKARA BIO INC URL http www takara bio com Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da Safety amp Handling of Adeno Associated Virus Vectors The protocols in this User Manual require the handling of adeno associated virus vectors It is im perative to fully understand the potential hazards of and necessary precautions for laboratory use of these vectors Viruses produced with AAV based vectors could depending on your gene insert be potentially hazardous Similar vectors have been approved for human gene therapy trials attesting to their potential ability to express genes in vivo For these reasons due caution must be exercised in the production and handling of any recombinant viruses Follow all applicable guidelines for rese
6. d titer of AAV particles are important factors for obtaining efficient and stable gene transfer in cultured cells and individual animals Cesium chloride density gradient centrifuga tion and iodixanol ultracentrifugation are conventional methods used to purify AAV particles However these methods are time consuming and require specialized equipment and advanced techniques The AAVpro Purification Kit All Serotypes can be used to easily purify AAV particles from AAV producing cells in about 4 hours Can be used for any AAV serotype High purity and recovery No complicated techniques such as ultracentrifugation Contains all buffers required to purify AAV particles from producer cells TAKARA BIO INC URL http www takara bio com Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da AAV producing cell Q AAV particle Oo Extraction of AAV particles using Extraction Solution A plus and B Treatment with Cryonase lt Cold active Nuclease Treatment with Precipitator Filtration by Millex HV 0 45 um filter Concentration using Amicon Ultra 15 filter WW Collection of AAV particle solution Figure 1 Purification of AAV particles using AAVpro Purification Kit All Serotypes URL http www takara bio com TAKARA BIO INC Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da Ill Components This product contains buffers and columns to perform four AAV purifications from packag
7. ing cells in five T225 flasks e ND ON Coc ae IV Storage AAV Extraction Solution A plus 22 ml x2 AAV Extraction Solution B 4 5 ml Precipitator A 5 ml Precipitator B 2 5 ml Millex HV 0 45 um 4 Amicon Ultra 15 100 kDa 4 Suspension Buffer 60 ml x 2 Cryonase Cold active Nuclease 500 ul Precipitator A may produce a white precipitate at low temperatures however this does not affect the quality or performance of this reagent If a precipitate is present dissolve completely at 37 C before use Cryonase Cold active Nuclease 20 C All other components Room temperature V Material Required but not Provided General equipment required for cell culture Syringe 0 5 M EDTA pH 8 0 e g EDTA Buffer Powder pH 8 0 Cat T9191 TAKARA BIO INC URL http www takara bio com Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da VI Protocol The protocol below describes purification of AAV particles from producer cells five T225 flasks We recommend using the AAVpro Helper Free Systems Cat 6230 6650 6663 for the prepara tion of AAV producing cells Not all products are available in all regions Please confirm availability in your region VI 1 Preparation of AAV extract solution 1 10 Detach the AAV producing cells by adding 1 80 volume of 0 5 M EDTA pH 8 0 to the culture medium incubate at room temperature for 10 min Collect the cells from the flask Centrifuge at 1
8. mly by pipetting Centrifuge at 2 000 X g for 5 min at 15 C and then confirm that the AAV solu tion inside the filter is lt 1 5 ml Note If the volume of the solution is gt 1 5 ml continue to centrifuge Repeat step VI 2 6 4 times total 5 times to obtain an appropriate volume of solution After discarding the filtrate resuspend the solution inside of the cup of the Amicon Ultra 15 100 kDa filter unit by pipetting or vortexing for 30 sec and transfer the solution to a new tube TAKARA BIO INC URL http www takara bio com Cat 6666 v201508Da AAVpro Purification Kit All Serotypes VII Experimental Examples VII 1 Purity of each AAV serotype after purification AAV particles carrying the fluorescent protein ZsGreen1 were prepared from producer cells in five T225 flasks The AAV solution was purified using this product and then titer was measured using the AAVpro Titration Kit for Real Time PCR Ver 2 Cat 6233 SDS PAGE was performed using 1 x 109 vector genome vg per lane The AAV capsid proteins VP1 VP2 and VP3 were confirmed to be the major bands present AAV Serotype M 1 2 3 5 6 n gt lt VP1 ae lt VP2 r EC lt VP3 ao M Protein Molecular Weight Marker Broad Cat 3452 URL http www takara bio com TAKARA BIO INC E Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da VII 2 Infectivity of purified AAV particles The infectivity of the AAV particles ob
9. nts All trademarks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions URL http www takara bio com TAKARA BIO INC
10. tained above was evaluated Cells were infected with purified AAV vectors at 5 000 vg cell serotypes 1 2 3 and 6 or 50 000 vg cell serotype 5 Flow cytometry analysis was performed 3 days later to determine infection efficiency The AAV particles purified using this kit were able to infect various cell lines _ AAV1 u 100 S 80 3 40 2 5 20 5 w w 5 si IN CHO RD HT1080 N RD HT1080 AAV3 100 80 60 40 20 0 CHO RD HT1080 HT1080 ZsGreen positive cells ZsGreen positive cells CHO RD AAV6 80 60 40 20 0 CHO RD HT1080 ZsGreen positive cells mS TAKARA BIO INC URL http www takara bio com Cat 6666 Takaka AAVpro Purification Kit All Serotypes v201508Da VIII Related products AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAVpro Helper Free System AAV2 2xU6 Cat 6661 AAVpro Helper Free System AAV5 2xU6 Cat 6662 AAVpro Helper Free System AAV6 2xU6 Cat 6663 pAAV ZsGreen1 Vector Cat 6231 AAVpro Packaging Plasmid AAV2 Cat 6234 AAVpro Packaging Plasmid AAV
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