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琼脂糖凝胶电泳
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==操作== # 参考实验视频:https://www.bilibili.com/video/BV1UR4y1x7ng/ # 制胶: ## 向锥形瓶中加入适量琼脂糖干粉和TAE电泳缓冲液50mL(Tris,乙酸,EDTA) ## 加热溶解,可超声or加磁力搅拌子煮沸 ## 冷却至60℃,冷却过程中摇晃or搅拌,避免底部先凝固,并及时挑出液面的凝胶膜和气泡 ## 冷却至不烫手后加入核酸染料(DNA goldview,注意避光),混匀,倒入凝胶模具(约厚3~5mm),挑去气泡或将气泡引至上样孔对侧。 [[File:凝胶模具示意图.jpg|center|thumb|琼脂糖凝胶模具示意图]] ## 室温冷却凝固(约30 min),拔出梳子,将凝胶及托盘转移至电泳槽中。向电泳槽中加入电泳缓冲液(约没过凝胶 2-3mm)。 ##加样: ## 样品:PBS or 超纯水稀释DNA样品至约50 ug/mL,吸取载样缓冲液(loading Buffer,LB,含有溴酚蓝)和样品混匀并上样10 uL至加样孔2,3…(上样量:8孔梳载样20 ul,16孔梳载样10ul) ## DNA Marker:含有不同分子量的标准品,可省略添加LB步骤。加载至gel加样孔(可置于开头、末尾、中间孔)。 #电泳 ##电泳槽置于冰水浴中,利于DNA样品稳定。 ##DNA含有磷酸基团带负电荷,由负极向正极泳动,150 V,负极出现气泡,电压可中途调整。 [[File:琼脂糖凝胶电泳示意图.jpg|center|thumb|琼脂糖凝胶电泳示意图]] ##12-15 min,一般溴酚蓝迁移至gel约2/3处停止电泳,取出电泳槽中的凝胶及托盘。 #成像 ##使用314的BioRad凝胶成像系统,image lab软件 ##从托盘中取出凝胶放置于样品盘中,选择通道<gold>,调整滤光片,观察显色。 [[File:琼脂糖凝胶结果图.png|center|thumb|琼脂糖凝胶结果图]]
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