银染聚丙烯酰胺凝胶电泳技术体系的优化

doi:10.6048/j.issn.1001-4330.2019.12.018

新疆农业科学 ›› 2019, Vol. 56 ›› Issue (12): 2312-2319.DOI: 10.6048/j.issn.1001-4330.2019.12.018

• 农产品安全·植物保护·畜牧兽医 • 上一篇下一篇

银染聚丙烯酰胺凝胶电泳技术体系的优化

王露露¹, 刘欣婷¹, 周涛¹, 王娟², 贾东海², 李强², 侯献飞², 石必显², 顾元国², 兰海燕¹

1.新疆大学生命科学与技术学院/新疆生物资源基因工程重点实验室,乌鲁木齐 830046;
2.新疆农业科学院经济作物研究所,乌鲁木齐 830091

收稿日期:2019-07-29 出版日期:2019-12-20 发布日期:2019-12-27
通信作者: 兰海燕(1969-),女,新疆人,教授,博士生导师,研究方向为植物抗逆分子生物学,(E-mail)lanhaiyan@xju.edu.cn
顾元国(1981-),男,新疆人,助理研究员,硕士,研究方向为油菜遗传育种与栽培生理,(E-mail)86322533@qq.com
作者简介:王露露(1995-),女,新疆人,硕士研究生,研究方向为植物抗逆分子生物学,(E-mail) 1264691915@qq.com
基金资助:
科技厅项目“南疆一年两熟区域麦后复种油菜的多元化利用与提质增效”(2019012A12);国家重点研发计划项目“长江上游及北方油菜高产优质适宜机械化新品种培育”(2018YFD0100500)

Optimization of Silver Staining Polyacrylamide Gel Electrophoresis Technology System

WANG Lu-lu¹, LIU Xin-ting¹, ZHOU Tao¹, WANG Juan², JIA Dong-hai², LI Qiang², HOU Xian-fei², SHI Bi-xian², GU Yuan-guo², LAN Hai-yan¹

1.Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China;
2.Institute of Economic Crop, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2019-07-29 Online:2019-12-20 Published:2019-12-27
Correspondence author: LAN Hai - yan (1969-), female, native place: Xinjiang, Professor, research field: Molecular biology of plant in stress tolerance, (E-mail) lanhaiyan@xju. edu.cn
GU Yuan - guo (1981-), male, native place: Xinjiang, Research fellow, research field: Oil seed rape varieties genetic breeding and cultivation physiology, (E-mail)86322533@qq.com

摘要/Abstract

摘要： 【目的】针对银染PAGE实验中常出现的背景颜色深、条带弥散、凝胶变形及条带不整齐等问题进行归纳总结并提出优化方法。【方法】以甘蓝型油菜基因组DNA为研究对象,通过PAGE电泳后银染并调整试剂配方、实验条件,筛选出适合实验的技术体系。【结果】甲醇浓度、电压稳定性、电泳温度、气泡等显著影响实验结果。甲醛浓度过高(1.25%)时,虽所需染色时间短,但凝胶背景深;甲醛浓度过低(0.001 25%)时,背景浅但条带淡,需要长时间染色;电压不稳定会造成条带弥散;电泳温度过高,导致凝胶变形,条带弯曲;凝胶中的气泡会扰乱DNA条带的形状;NaOH浓度在0.875%~1.375%,染色结果无明显差异。【结论】甲醛用量4 mL(1%)、保持稳定电压、在冰浴中电泳、制胶时及时赶走所有气泡,可得到凝胶背景浅、条带清晰且正常的结果。

关键词: 聚丙烯酰胺凝胶电泳; 银染; 弥散现象; 体系优化

Abstract: 【Objective】 In the present study, the problems of gel background was too dark, band smear, the gel was distorted and DNA bands were bent in silver staining PAGE experiments were summarized and the method was optimized. 【Method】Genomic DNA of Brassica napus L. was employed as material, the reagent dosage and experimental conditions of PAGE electrophoresis and silver staining technique were adjusted to obtain the best effects. 【Result】Results showed that the staining effects were interfered by different factors, e.g. methanol concentration, voltage stability, electrophoresis temperature, bubbles trapped in gel and other issues. When methanol concentration was too high (1.25%), the gel background was too dark although staining in short time; if it was too low (0.001 25%), gel background was light but DNA bands were light too, and much longer time was needed to complete staining; unstable voltage could result in bands smear; when the electrophoresis temperature was too high, the gel was distorted and DNA bands were bent; bubbles in gel could disturb the shape of DNA bands. However, there was no significant difference in staining effect by adjusting NaOH concentration from 0.875% to 1.375%.【Conclusion】A modified silver staining PAGE method was concluded, which was improved as: 4 mL methanol (1%), stable voltage, electrophoresis in ice bath, and driving all bubbles in time in experimental system, and then a clear background with smooth, distinct bands could be obtained.

Key words: polyacrylamide gel electrophoresis; silver staining; band smear; system optimization

中图分类号:

S188

王露露, 刘欣婷, 周涛, 王娟, 贾东海, 李强, 侯献飞, 石必显, 顾元国, 兰海燕. 银染聚丙烯酰胺凝胶电泳技术体系的优化[J]. 新疆农业科学, 2019, 56(12): 2312-2319.

WANG Lu-lu, LIU Xin-ting, ZHOU Tao, WANG Juan, JIA Dong-hai, LI Qiang, HOU Xian-fei, SHI Bi-xian, GU Yuan-guo, LAN Hai-yan. Optimization of Silver Staining Polyacrylamide Gel Electrophoresis Technology System[J]. Xinjiang Agricultural Sciences, 2019, 56(12): 2312-2319.

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银染聚丙烯酰胺凝胶电泳技术体系的优化

Optimization of Silver Staining Polyacrylamide Gel Electrophoresis Technology System

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