Protocols
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Immunohistochemistry(IHC) ProtocolProtocols 2023. 6. 15. 22:19
Protocol *All in RT, shaking at 35 rpm *24-well-plate standard 1. Wash with 0.3% PBS-T, 5 min x 3 (500ul/well) * PBS-T: 0.1M PBS 1L + TritonX-100 3ml 2. Blocking for 1.5hr (40~90min) (500ul/well) * Blocking solution: - 0.15% PBS-T 15ml - BSA 0.3g - NDS(normal donkey* serum) 750ul *depends on the host of secondary antibody used 3. 1st Ab attachment O/N (16~20hr) (250ul/well) - Blocking solution :..
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Basic Cell Work ProtocolProtocols 2023. 6. 13. 23:35
Protocol Ⅰ. Cell work Media 만들기 *Materials: DMEM 500ml x2, Anti-biotics, 10X FBS 1. DMEM 10ml 빼서 버리기 2. A-A 10ml 넣어주기 3. FBS 100ml 추가한 후 filtering* *media 부어서 새지 않는 것 확인한 뒤 suction기 틀어주기 Ⅱ. Cell thawing 1. 질소탱크에서 Cell stock 꺼낸 뒤 37℃ water bath에서 녹여주기 2. 15ml tube에 차가운 media 9ml 넣어주기 3. Cell stock vial에서 1ml 딴 뒤 15ml tube에 넣어주기 *딸 때 약하게 pipetting, 15ml tube에 넣어줄 때도 마찬가지 4. Centrifuge for 5 min, 1..
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RT-qPCR (Real-Time quantitative PCR) protocolProtocols 2023. 4. 13. 23:45
Protocol Ⅰ. RNA extraction 1. RLT buffer 600ul(pellet 양에 따라 조절) pellet에 넣고 homogenization 시켜주기 * Lysis(RLT) buffer 1ml 당 β-mercaptoethanol(b-ME) 10ul 섞어서 사용 2. 동량의 70% EtOH 넣은 뒤 mixture를 column으로 옮겨주기 3. Centrifuge 1min 4. RW1, column 당 700ul 넣고 centrifuge 1min 5. RPE buffer, column 당 500ul, centrifuge 1min 6. RPE buffer, column 당 500ul, centrifuge 2min 7. (optional) centrifuge in full speed(ove..
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Genotyping protocolProtocols 2023. 4. 13. 22:40
protocol 1. Tail lysis *Get the tissue sample from the mouse tail or ear *Tail lysis buffer(TLB) composition (500ml standard) - 1.5M Tris (pH 8.8) 33.3ml - 4M NaCl 25ml - 0.5M EDTA 5ml - 10% SDS 10ml - DW 1) Mix Proteinase-K with Tail lysis buffer [proteinase-K: TLB = 1:100] 2) Put 300ul of the mixture in each ep-tube with a tail in it. 3) O/N in 60℃ 2. DNA extraction 1) Put 700ul of pure EtOH i..
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Western Blot ProtocolProtocols 2023. 4. 13. 21:28
Protocol Ⅰ. Sampling 1. Protein preparation in ice 2. Bradford Protein Assay 1) 5X Bradford dilution with auto DW 3) 96 well plate에 1X Bradford 넣기 for sample: 200ul/well for standard curve: prepare serial dilution (5 well: 200ul/well, 1 well: 400ul/well) 4) BSA serial dilution: 2mg/ml BSA 4ul in 400ul Bradford well (highest concentration: 20ug/1ml ) 5) Lysate 2ul씩 각 well에 넣은 뒤 잘 섞이도록 pipetting 6..