《GUS组织化学染色(9页).doc》由会员分享,可在线阅读,更多相关《GUS组织化学染色(9页).doc(9页珍藏版)》请在得力文库 - 分享文档赚钱的网站上搜索。
1、-GUS组织化学染色1. 实验原理适宜的反应条件下,-葡聚糖苷酶(GUS)可将X-Gluc水解成蓝色物质,因此具有GUS活性的部位或位点呈现蓝色或蓝色斑点,可用肉眼或显微镜观察到。2 实验材料1)转基因烟草叶片或试管苗2)非转基因烟草的叶片或试管苗(阴性对照)3 药品试剂1)X-Gluc,用N-N-二甲基酰胺配成20mM的贮存液,分装成每管100L,保存于-20。2)底物溶液(染色液):3)1mM X-Gluc加入100mM磷酸钠缓冲液(pH=7.0),缓冲液中含10mM EDTA-Na2,1-5mM K3Fe(CN)6(铁氰化钾),1-5mM K4Fe(CN)6(亚铁氰化钾),0.001%(
2、V/V)Triton X-1004 实验器材小药瓶,培养皿,培养箱(37),微量移液器5 实验步骤1)将准备好的叶片放入小药瓶,加入染色液浸没试材,封好盖子;2)37培养箱中温育1小时至过夜;3)将浸染过的试材转入70%或95%乙醇中脱色2-3次(除去叶绿素),至阴性对照材料呈白色为止。6 结果叶片上GUS活性的部位或位点呈现蓝色或蓝色斑点。 先用DMSO溶解X-GLUC 好像是100mg/2ml PBF 94ML+X-GLUC 0.1ML+TRITION X-100 2ML +FE2+ 2ML+ FE3+ 2ML Gus staining 1. Excise root and leaf se
3、gments, wash with 100mM potassiumphosphate buffer (pH7.0) for 3 times.2. Immerse in the Gus substrate solution, 37C, dark, 12-24h3. Rinse in phosphate buffer4. Fix for 4h or longer5. Rinse in the same buffer6. Observe as whole specimens or as sections.1M potassium phosphate buffer (pH7.0)K2HPO4: 34.
4、8g -200mlKH2PO4: 27.2g-200ml184.5ml K2HPO4+115.5ml KH2PO4- 300ml 1M potassium phosphate buffer(pH7.0)Gus substrate solutionVolume(500ml)Final concentrationPotassium ferricyanide 82mg0.5mMPotassium ferrocyanide105.6mg0.5mMPotassium phosphate buffer (pH7.0, 1M)50ml100mMStore at -20C. Add 1mM x-gulc (M
5、W521.80.5mg/ml) freshly when use.Fix solution2.5% glutaraldehyde200mM sodium cacodylate, pH7.2 共培养后愈伤的瞬时表达、抗性愈伤的鉴定以及转基因植株的鉴定用GUS组织化学法测定.将待测定的材料,如愈伤或叶片,浸入适量X-Gluc溶液,于37 保温过夜后,在体视显微镜下观察,拍照记录.若将加有X-Gluc溶液的材料抽真空,染色效果将更好.如材料存在色素干扰问题,染色后用酒精脱色,使色素的颜色褪掉而使染成的蓝色保存.X-Gluc染色液:(20*)0.2mol/LNa3PO4缓冲液(62mL0.2mol/L
6、Na2HPO4;38mL0.2mol/LNaH2PO4),pH7.0; 0.1 mol/L K3Fe(CN)6;0.1 mol/L K4Fe(CN)6.3H2O;1.0 mol/L Na2EPTA;0.1% X-Gluc. 1000mlx-gluc 1000mg (20ml dmso)Na2HPO4: 620*0.2*M=0.620*0.2*358(12H2O)=44NaH2PO4: 380*0.2*M=0.358*0.2*156(2H2O)=11 K3Fe(CN)6:1000*0.01*M=32926*0.01=3.3 K4Fe(CN)6:1000*0.01*M=42239(3H2O)*0.
7、01=4.2Triton 1MLNa2EPTA:1000*0.1*M=372.24(2H2O)=37 1. 取5 mg X-Gluc (5-bromo-4-chloro-3-indolyl glucuronide)溶於100 l DMSO於中. 2. 加入10 ml reaction buffer (10 mM Na2EDTA, 100 mM NaH2PO4H2O, 0.1% Triton, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, pH 7.0). 3. 混合均匀后分装於eppendorf tube中,保存於-20冰箱内. 100ml x-gluc染液配制x-gluc 50mg加入溶入1ml dmso1ml triton 100Na2EDTA (20mM) 10mM 5ml ; K3Fe(CN)6 (0.1M) 5mM 0.5mlK4Fe(CN)6 (0.1M) 5mM 0.5ml ; Na2HPO4 (0.2M) 100mM 3.1mlNaH2PO4 (0.2M) 100mM 1.9ml X-Gluc 0.1% 10ul第 9 页-
限制150内