Roche 11745832910 DNA高效地高辛标记及检..._标记试剂盒_其它试剂盒_试剂盒_知道

DIG-DNA标记与检测

一、探针DNA标记方法

步骤：
1．10ng~3ugDNA每管，双蒸水定量至总体积15ul
2．沸水水浴10分钟后迅速冰浴
3．加入 5号试剂 2ul ， 6号试剂 2ul ，7号试剂 1ul
4．37度1小时~20小时
5．中止反应加2ul 0.2M EDTA (pH 8.0) 和/或 65度水浴 10分钟

二、标记效率检测
（一）试剂配置
Solution Composition Preparation
Washing buffer 0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20° C); 0.3% (v/v) Tween 20
Maleic acid buffer 0.1 M Maleic acid, 0.15 M NaCl; adjustwith NaOH (solid) to pH 7.5 (20° C)
Detection buffer 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20° C)

TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 8.0

Blocking stock solution Dissolve Blocking reagent (bottle 10) 10% (w/v) in Maleic
(10 × conc.) acid buffer underconstantly stirring on a heating block(65°C) or heat in a microwave oven,autoclave. The solution remains opaque

Blocking solution Prepare a 1 × working solution by dilutingthe 10 × Blocking solution 1:10in Maleic acid buffer.

Antibody solution Centrifuge Anti-Digoxigenin-AP(vial 8) for 5 min at 10 000 rpm in theoriginal vial prior to each use, and pipet the necessary amount carefully from thesurface. Dilute Anti-
Digoxigenin-AP 1: 5 000 (150 mU/ml) in Blocking solution.

Colorsubstrate Add 40 _l of NBT/BCIP (vial 9) to 2 ml of Detection buffer.
solution Note: Store protected from light!

（二）对照的标记DNA（4号试剂）系列稀释

（三）步骤
1、取以上制备的管2~9各1ul，以及自己标记的探针1ul，点到一小片尼龙膜
2、通过紫外线或者120度半小时使核酸交连到膜上
3、膜置塑料盒中加Maleic acid buffer 20ml ，15~25度轻摇孵育2分钟
4、10ml Blocking solution 孵育30分钟
5、10ml Antibody solution 孵育30分钟
6、10ml Washing buffer 洗2次，每次15分钟
7、10ml Detection buffer中平衡2~5分钟
8、膜在2ml新鲜配制的 Colorsubstrate solution 中避光孵育。显色期间避免摇动
9、中止反应用TE-buffer或者双蒸水洗5分钟

蛋白酶K预处理
三、样品检测与杂交
（一）步骤
1、稀释供试品及阳性对照DNA，点膜
2、通过紫外线或者120度半小时使核酸交连到膜上
3、将标记好的探针稀释到约25ng/ml, 煮沸5分钟后迅速冰浴
4、膜放入预热好的预杂交液（3.5ml/100cm2）中，充分混匀，避免起泡
5、倒掉预杂交液，加入杂交液及已变性的探针。杂交温度下至少孵育6小时

四、洗膜
1、2 × SSC, 0.1% SDS ， 15-25° C，洗膜2次，每次5分钟。
2、0.5× SSC, 0.1% SDS ，预热至65~68° C，洗膜2次，每次5分钟

五、结果检测
（一）试剂配制
Solution Composition Preparation
Washing buffer 0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20° C); 0.3% (v/v) Tween 20
Maleic acid buffer 0.1 M Maleic acid, 0.15 M NaCl; adjustwith NaOH (solid) to pH 7.5 (20° C)
Detection buffer 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20° C)

TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 8.0
Blocking stock solution Dissolve Blocking reagent (bottle 10) 10% (w/v) in Maleic
(10 × conc.) acid buffer underconstantly stirring on a heating block(65°C) or heat in a microwave oven,autoclave. The solution remains opaque

Blocking solution Prepare a 1 × working solution by dilutingthe 10 × Blocking solution1:10in Maleic acid buffer.

Antibody solution Centrifuge Anti-Digoxigenin-AP(vial 8) for 5 min at 10 000 rpm in theoriginal vial prior to each use, and pipet the necessary amount carefully from thesurface. Dilute Anti-
Digoxigenin-AP 1: 5 000 (150 mU/ml) in Blocking solution.

Colorsubstrate Add 40 _l of NBT/BCIP (vial 9) to 2 ml of Detection buffer.
solution Note: Store protected from light!

（二）步骤
1、Washing buffer洗膜1~5分钟
2、100ml Blocking solution 孵育30分钟
3、20ml Antibody solution 孵育30分钟
4、100ml Washing buffer 洗2次，每次15分钟
5、20ml Detection buffer中平衡2~5分钟
6、膜在10ml新鲜配制的 Colorsubstrate solution 中避光孵育。显色期间摇动
7、中止反应用TE-buffer或者双蒸水50ml洗5分钟展开

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Roche 11745832910 DNA高效地高辛标记及检...