VIRAL VECTOR PLATFORM SERVICE RETROVIRUS* APPLICATION FORM (REG-VVB-PVV-PNT-001-02.01) BILLING REQUESTER: PI: INSTITUTION: ADDRESS: PHONE NUMBER: PI's e-MAIL: DELIVERY (do not fill in if the same as above) NAME: INSTITUTION: SHIPPING ADDRESS: PHONE NUMBER: e-MAIL: Please fill the data below and send the document by e-mail to Naiara Tejados(1) NUMBER OF BATCHES (2) Mini-Retro: Supernatant from 1 P100 packaging-cell transfection Midi-Retro: Supernatant from 1 P150 packaging-cell transfection Maxi-Retro: Supernatant from 3 P150 packaging-cell transfection Note: If you need more than one viral vector separate the data of each one with // TYPE OF VIRAL VECTOR Shuttle plasmid (name, designation) Length (in Kbp) of the packable construct Transgene (type, special features if any) Reporter gene Promoter Self-inactivating Yes Pseudotype VSVg * No ampho eco P101A1 NOTICE: Nowadays retroviruses are not the most frequently chosen viral vectors. Producer cells are commonly stored frozen, and thawing of the producer cells is followed by quality control testing, and must be performed for almost every order. VIVEbioTECH - Parque Científico y Tecnológico de Gipuzkoa // P. Mikeletegi 81 // 20009 - San Sebastián (Gipuzkoa) País Vasco (Spain) www.vivebiotech.com // info@vivebiotech.com // Telf. 943308568 // Fax. 943308222 DNA PURITY OF SHUTTLE PLASMID (PROVIDE BY USER) (3) 260/280 Volume Concentration TITRATION (4) (5) Titration by default Transduction units (T.U./mL) Viral particles containing genome (v.g./mL) Titration on request (at an additional cost) p24 ELISA Viral inserted copies (inserts/cell) CONCENTRATION Concentration using PEG (available for Mini-Retro and Midi-Retro) Ultracentrifugation (available for Maxi-Retro) VIVEbioTECH - Parque Científico y Tecnológico de Gipuzkoa // P. Mikeletegi 81 // 20009 - San Sebastián (Gipuzkoa) País Vasco (Spain) www.vivebiotech.com // info@vivebiotech.com // Telf. 943308568 // Fax. 943308222 ADDITIONAL NOTES: 1. For any additional information or comments please contact Naiara Tejados (send), who will pass on any doubts and additional information requests to the production personnel. 2. SCALE OF PRODUCTION Producer cells: GP2.293. Supernatant per P150 dish: 20 mL. Mini-Retro: 1 P100 supernatant. Batches cannot be concentrated by ultracentrifugation. Clarified supernatant will be delivered. Vectors can be concentrated using PEG on request. Midi-Retro: 1 P150 supernatant. Batches cannot be concentrated by ultracentrifugation. Clarified supernatant will be delivered. Vectors can be concentrated using PEG on request. Maxi-Retro: 3 P150 supernatant. Vectors can be concentrated by ultracentrifugation on request. 3. QUALITY of DNA. Minimum purity values 260/280=1.8. RNA free. A photo of an EtBr, or similarly stained agarose gel will be required (attach as pdf or jpg format). Competent Stbl3 or similar, and Endo-free DNA purification columns are strongly recommended. DNA must be resuspended in H2O at 1 mg/mL. 30 micrograms of DNA are needed to transfect one P150 dish. 4. TITRATION methods. ALL the batches produced at VIVEbioTECH will be titrated and data delivered. Depending on the specificities of the vectors, titrations will be done by default as follows: Whenever the transfer plasmid does encode for a fluorescent reporter that can be properly excited by the 488 or the 635 nm laser, ALL the batches will be titrated by transducing indicator HEK293T cells and FACS analysis. Unit: transduction units per mL (T.U./mL). These batches can also be titrated by qRT-PCR under request. Whenever the transfer plasmid does not encode for a fluorescent reporter, ALL the batches will be titrated by qRT-PCR. Unit: viral genomes per mL (vg/mL). Normally, following our procedures, 1 T.U. is equal to 300-500 viral-genome containing particles. For viruses bearing an antibiotic-resistant gene, ask about pricing. Additionally, titration by integration events by qPCR on genomic DNA or by p24 ELISA can be provided upon request at an additional cost. Final titers are regularly >5x105 T.U./mL for non-concentrated batches and >5x107 T.U./mL for concentrated stocks. 5. YIELDS. Final yields depend on the size and specificities of the shuttle plasmid and the transgene. IMPORTANT INFORMATION 1- All vectors delivered by the platform are for research use only. 2- The generated retrovectors need to be handled and used under BSL-II. VIVEbioTECH - Parque Científico y Tecnológico de Gipuzkoa // P. Mikeletegi 81 // 20009 - San Sebastián (Gipuzkoa) País Vasco (Spain) www.vivebiotech.com // info@vivebiotech.com // Telf. 943308568 // Fax. 943308222 GENERAL CONDITIONS 1. Retroviral stocks will be dispatched within one month following receipt of the transfer plasmid, under the conditions specified above. The customer will be notified of any delay due to justified but unforeseeable shortly after realisation. 2. The customer’s material will be safeguarded for a six-month period after the delivery of the viral stocks. Thereafter it will be destroyed. The customer will be informed of any incident regarding the quality, damage or loss of the material during storage at VIVEbioTECH, and this will be duly registered according to VIVEbioTECH Quality Guidelines. 3. VIVEbioTECH ensures the complete confidentiality for the whole process, for both the reagents and the materials from the customer’s side, and the produced viruses. 4. Production of retroviral vectors at VIVEbioTECH is based on the CLONTECH product RetroX Universal packaging Vector Set (Catalog No. 631457). We strongly encourage customers to refer to this product whenever data regarding the retrovirus is published or shared. 5. VIVEbioTECH would appreciate the inclusion of “VIVEBIOTECH SL” as the manufacturer of the retrovirus in any related publications or research projects. VIVEbioTECH - Parque Científico y Tecnológico de Gipuzkoa // P. Mikeletegi 81 // 20009 - San Sebastián (Gipuzkoa) País Vasco (Spain) www.vivebiotech.com // info@vivebiotech.com // Telf. 943308568 // Fax. 943308222