AFFYMETRIX基因芯片操作流程

1、A 生 B旦 C净 D 丑至 200l 至 300l 终体积100l200l300l20×Eukaryotic Hybridization Controls 冻存，使用前 65， 5 分钟2.使用前室温平衡探针3.99， 5 分钟4.通过加样孔加入适量体积（见表2.2）1×Hybridization Buffer 湿润芯片5. 45， 60rpm 预杂交芯片 10 分钟6. 步骤 3 处理过的样品 45， 5 分钟7. 最大速离心 5 分钟8. 从芯片中取出 Buffer ，加等体积处理好的杂交液9. 45， 60rpm 杂交芯片 16 小时表 2.2Array杂交体积总体

2、积Standard200l250lMidi130l160lMini80l100lMicro80l100l第三章 洗脱、染色、扫描芯片<一>实验和洗涤工作站设置一、进入 Experiment Information洗脱、染色、扫描芯片之前都必须先定义一个Experiment二、准备洗涤工作站基因芯片洗涤工作站400 用来洗脱和染色探针阵列。<二>洗脱和染色1.杂交 16 小时后，从芯片中取出杂交液装入一个新的离心管，放置冰上或 -80长时间保存。2. Wash Buffer A 充满芯片3.配制下列溶液表 3.1 SAPE 液（使用前配制， 4储存）成分体积终浓度2

3、15;MES Stain Buffer600.0 l1×50mg/ml acetyed BSA48.0 l2mg/ml1mg/ml Streptavidin-12.0 l10 g/mlPhycoerythrin(SAPEDI 水540.0 l总体积表 3.2 抗体溶液成分2×MES Stain Buffer50mg/ml acetyed BSA10mg/ml Normal Goat IgG0.5mg/ml biotinylated antibodyDI 水总体积4.洗涤工作站按表3.3 工作表 3.31200l体积终浓度300.0 l1×24.0 l2mg/ml6

4、.0 l0.1mg/ml3.6 l3 g/ml266.4 l600l标准格式EukGE-WS2Post Hyb 10 cycles of 2 Wash#1 mixes/cycle withWash Buffer A at25Midi 格式Midi-euk210 cycles of 2 mixes/cycle with Wash Buffer A at 30 Micro/Mini 格式Micro-1v1/Mini-euk210 cycles of 2 mixes/cycle with Wash Buffer A at 25 Post Hyb 4 cycles of 15 Wash#2 mixes

5、/cycle withWash Buffer B at50StainStain the probe arrayfor 10minutes inSAPE solution at25Post Stain 10 cycles of 4Washmixes/cycle with Wash Buffer A at 252nd Stain Stain the probe array for 10minutes in antibody solution at 253nd Stain Stain the probe array for 10minutes in SAPE solution at 25Final1

6、5 cycles of 4Washmixes/cycle withWash Buffer A at30The holdingtemperature is 25<三>扫描6 cycles of 15 mixes/cycle with Wash Buffer B at 50Stain the probe array for 5minutes in SAPE solution at 3510 cycles of 4 mixes/cycle with Wash Buffer A at 30 Stain the probe array for 5minutes in antibody sol

7、ution at 35Stain the probe array for 5minutes in SAPE solution at 3515 cycles of 4 mixes/cycle with Wash Buffer A at 35 The holding temperature is 258 cycles of 15 mixes/cycle with Wash Buffer B at 50Stain the probe array for 10minutes in SAPE solution at 2510 cycles of 4 mixes/cycle with Wash Buffe

8、r A at 30 Stain the probe array for 10minutes in antibody solution at 25Stain the probe array for 10minutes in SAPE solution at 2515 cycles of 4 mixes/cycle with Wash Buffer A at 35 The holdingtemperature is 25<四 >关闭洗涤工作站附：溶液配制5×RNA Fragmentation Buffer:(200mM Tris-acetate,PH 8.1,500mM KO

9、Ac,150mM MgOAc4.0ml 1M Tris-acetate,PH 8.1(冰醋酸调节 PH0.64g MgOAc0.98g KOAcDEPC 处理水至 20ml强烈搅拌，混合均匀0.2 m过滤室温保存12×MES Stock：+(1.22M MES,0.89MNa 70.4g MES free acid monohydrate193.3g MES Sodium Salt800ml 分子生物级水混合，定溶至 1000ml. PH 在 6.5-6.7 之间0.2 m过滤不灭菌， 2-8避光保存2×Hybridization Buffer:(1 ×:100m

10、M MES, 1M Na +,20mM EDTA,0.01%Tween2 50ml:8.3ml 12 MES× Stock17.7ml 5M NaCl4.0ml 0.5M EDTA0.1ml 10%Tween2019.9ml 水2-8避光保存Wash Buffer A：（ 6×SSPE,0.01%Tween20）1000ml：300ml 20 ×SSPE1.0ml 10%Tween20698ml 水0.2 m过滤Wash Buffer B：（ 100mM MES,0.1M Na +,0.01%Tween20）1000ml：83.3ml 12 MES× Stock Buffer5.2ml 5M NaCl1.0ml 10%Tween20910.5ml 水0.2 m过滤2-8避光保存2×Stain Buffer：(1 ×:100m