Protocols
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AAV Purification & Concentration ProtocolProtocols 2025. 2. 15. 18:10
* (D1556-250Ml) OptiPrep™ Density Gradient Medium by Sigma is 60% Iodixanol by W/V* The following volumes are calculated for the rotor (Beckman-Coulter VTI 70) using 32 ml OptiSeal tubes (Cat # 361625) * 1 M NaCl/PBS-MK buffer - Dissolve 5.84 g of NaCl, 26.3 mg of MgCl2and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. - Sterilize by passing through a 0.22-μm filter and store at 4 ℃.*..
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AAV Production ProtocolProtocols 2025. 2. 15. 18:06
1. Timeline- Day1: Cell seeding- Day2: Cell transfection- Day5: Cell harvest & lysis 2. Protocol Day 1 Seed HEK293T cells with DMEM/10%FBS/1%a.a medium at each dish* 10cm dish: 4.0x106 cells 15cm dish: 9.0x106 cells* Confluence at 70-80% next morning Day 2 Proceed transfection as below. * PEI solution (1ug/ul)- Dissolve ~100mg of branched PEI solution in ~10ml of DDW.- Dilute at 10..
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Midi-prep ProtocolProtocols 2023. 8. 30. 20:55
Protocol * Materials - Midi-prep Kit: MN midi-prep kit - 세포 배양 적당량: 100~200ml - LB broth 1) Dissolve LB-Broth (25g/L) in DW and autoclave 2) Leave the solution at RT to cool down 3) Add Ampicillin at a concentration of 100ug/ml just before using * Methods Ⅰ. Midi-culture 1. Transformation이 완료된 LB plate에서 colony harvest, 100ml LB broth에 넣어주기 2. 37℃ Incubator에서 O/N culture, shaking Ⅱ. Midi-prep 1...
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Mini-prep ProtocolProtocols 2023. 8. 27. 20:23
Protocol * Materials - Mini-prep Kit: QIAGEN, spin column - 세포 배양 적당량: ~10ml - LB broth 1) Dissolve LB-Broth (25g/L) in DW and autoclave 2) Leave the solution at RT to cool down 3) Add Ampicillin at a concentration of 100ug/ml just before using * Methods - Transformation이 완료되어 생긴 colony 하나를 LB broth 에서 O/N 배양 (37℃, shaking) 1. 2ml tube에 배양액 1.9m씩 넣어주기 2. Centrifuge 6000 rpm, 1 min, 4℃, supernata..
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Transformation ProtocolProtocols 2023. 8. 27. 20:19
Protocol * Materials - Competent cell (DH5α) - Ampicillin - LB plate 1) Dissolve LB-Agar (40g/L) in DW and autoclave 2) Once the temperature drops to 45-50℃, add Ampicillin at a final concentration of 100ug/ml 3) Before the LB-Agar solution solidifies, pour 15~20ml each into the 90-100mm petri dish 4) Dry the plate upside-down, O/N 5) Store the plate in 4℃ * Methods * Make sure the LB plate is w..