GUS组织化学染色
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*)
0.2 mol/L Na3PO4缓冲液(62 mL 0.2 mol/L Na2HPO4;38 mL 0.2 mol/L NaH2PO4),pH 7.0; 0.1 mol/L K3[Fe(CN)6];0.1 mol/L K4[Fe(CN)6].3H2O;1.0 mol/L Na2EPTA;0.1% X-Gluc.
1000ml
x-gluc 1000mg (20ml dmso)
Na2HPO4: 620*0.2*M=0.620*0.2*358(12H2O)=44
NaH2PO4: 380*0.2*M=0.358*0.2*156(2H2O)=11
K3[Fe(CN)6:1000*0.01*M=329.26*0.01=3.3
K4[Fe(CN)6]:1000*0.01*M=422.39(3H2O)*0.01=4.2
Triton 1ML
Na2EPTA: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 NaH2PO4·H2O, 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 dmso
1ml triton 100
Na2EDTA (20mM) 10mM 5ml ; K3Fe(CN)6 (0.1M) 5mM 0.5ml
K4Fe(CN)6 (0.1M) 5mM 0.5ml ; Na2HPO4 (0.2M) 100mM 3.1ml
NaH2PO4 (0.2M) 100mM 1.9ml