2018. 5. 15. 15:05
Study/draft
- flow cell
- glass 준비 : 구멍 뚫린 것, 구멍 뚫리지 않은 것
- acetone sonication wash. 15분 이상
- dW wash & N2 air dry
- plasma cleaning 20분
- APTES coating(acteon 48 ml, dW 1ml, APTES 1ml) 10분 이상
- dW wash & N2 air dry
- para film 이용하여 flow cell 제작 및 진공포장하여 저장
- DNA construct
- transformation(pPIA 2-6 & pUC19) & preculture & mini/midi prep.
- linker PCR
- biotin-dUTP/DBCO-dUTP/Dig-dUTP, pUC19 template 이용하여 PCR
- enzyme cut
- pPIA2-6 : BamH1 & Sac1
- biotin-linker : BamH1
- DBCO-linker : Sac1
- enzymed DNA ligation
- flow cell chamber 제작 및 DNA 준비
- 10xPBS(pH7.4) from 3M 사용
- washing buffer(20 mM Tris, 5 mM EDTA, pH7.4)
- washing buffer(with 0.05% tween20)
- MS(PEG)4 and NHS-(PEG)4-Azide dissolved in DMSO (250mM)
- MS(PEG)4 and NHS-(PEG)4-Azide 각각 50mM 되도록 1xPBS로 dilution and flow cell insert, 1시간 이상 incubation(room temperature)
flow cell wash by washing buffer(20 mM Tris, 5 mM EDTA, pH7.4 )
flow cell에 tubing 연결하여 M.T.에 올림
DNA & m280 bead mixture washing buffer(with 0.05% tween20)로 pull down 하여 flow cell 삽입, 1시간 이상 incubation
1xPBS wash
- NAP1 준비
- 75.11 uM NAP1 aliquot 및 급속냉동(액체질소 사용)하여 -80도 냉동고 보관
- NAP1 Base buffer(50mM KCL, 25mM HEPES, 0.1mM EDTA, pH 7.56)
- 5% PEG & PvOH
- 40mg/ml BSA
- Incubation buffer
- base buffer : 925 ul
- 5% PEG & PvOH : 50 ul
- 40mg/ml BSA : 25 ul
- Exp. buffer
- base buffer : 992.5 ul
- 5% PEG & PvOH : 5 ul
- 40mg/ml BSA : 2.5 ul
- NH mixture incubation on ice 30분 이상
- incubation buffer : 91.5 ul
- 10uM NAP1 : 5ul
- 20uM H2A/B dimer : 2 ul
- 10uM H3.1/4 tetramer : 1.5ul
- NH mixturue 1/100 ~1/1000 dilution 하여 실험