琼脂糖凝胶电泳的操作步骤
琼脂糖凝胶电泳是用琼脂或琼脂糖作支持介质的一种电泳方法。对于分子量较大的样品，如大分子核酸、病毒等，一般可采用孔径较大的琼脂糖凝胶进行电泳分离。
琼脂糖凝胶约可区分相差100bp 的DNA 片段，其分辨率虽比聚丙烯酰胺凝胶低，但它制备容易，分离范围广，尤其适于分离大片段DNA。普通琼脂糖凝胶分离DNA 的范围为0.2-20kb，利用脉冲电泳，可分离高达10^7bp 的DNA 片段。
操作流程
准备干净的配胶板和电泳槽注意DNA 酶污染的仪器可能会降解DNA，造成条带信号弱、模糊甚至缺失的现象。
选择电泳方法
一般的核酸检测只需要琼脂糖凝胶电泳就可以；如果需要分辨率高的电泳，特别是只有几个bp 的差别应该选择聚丙烯酰胺凝胶电泳；用普通电泳不合适的巨大DNA 链应该使用脉冲凝胶电泳。注意巨大的DNA 链用普通电泳可能跑不出胶孔导致缺带。
正确选择凝胶浓度
对于琼脂糖凝胶电泳，浓度通常在0.5～2％之间，低浓度的用来进行大片段核酸的电泳，高浓度的用来进行小片段分析。低浓度胶易碎，小心操作和使用质量好的琼脂糖是解决办法。注意高浓度的胶可能使分子大小相近的DNA 带不易分辨，造成条带缺失现象。
适合的电泳缓冲液
常用的缓冲液有TAE 和TBE，而TBE 比TAE 有着更好的缓冲能力。电泳时使用新制的缓冲液可以明显提高电泳效果。注意电泳缓冲液多次使用后，离子强度降低，PH 值上升，缓冲性能下降，可能使DNA 电泳产生条带模糊和不规则的DNA 带迁移的现象。中国最大的汉奸
电泳的合适电压和温度
电泳时电压不应该超过20V/cm，电泳温度应该低于30℃，对于巨大的
DNA 电泳，温度应该低于15℃。注意如果电泳时电压和温度过高，可能导致出现条带模糊和不规则的DNA 带迁移的现象。特别是电压太大可能导致小片段跑出胶而出现缺带现象
DNA 样品的纯度和状态
注意样品中含盐量太高和含杂质蛋白均可以产生条带模糊和条带缺失的现象。乙醇沉淀可以去除多余
的盐，用酚可以去除蛋白。注意变性的DNA 样品可能导致条带模糊和缺失，也可能出现不规则的DNA 条带迁移。在上样前不要对DNA 样品加热，用20mM NaCl 缓冲液稀释可以防止DNA 变性。
DNA 的上样
正确的DNA 上样量是条带清晰的保证。注意太多的DNA 上样量可能导致DNA 带型模糊，而太小的DNA 上样量则导致带信号弱甚至缺失。TIANGEN 公司DNA 分子量标准每次上样6ul 即可得到清晰均匀的条带。Marker 的选择
DNA 电泳一定要使用DNA Marker 或已知大小的正对照DNA 来估计DNA 片段大小。Marker 应该选择在目标片段大小附近ladder 较密的，这样对目标片段大小的估计才比较准确。TIANGEN 公司的DNA Marker 条带清晰，亮度均匀，质量稳定，是您实验的首选。需要注意的是Marker 的电泳同样也要符合DNA 电泳的操作标准。如果选择λDNA/HindIII 或者λDNA/EcoRI 的酶切Marker，需要预先65℃加热5min，冰上冷却后使用。从而避免HindIII 或EcoRI 酶切造成的粘性接头导致的片段连接不规则或条带信号弱等现象。
凝胶的染色和观察
实验室常用的核酸染色剂是溴化乙啶（EB），染色效果好，操作方便，但是稳定性差，具有毒性。而
海马煲汤其他系列例如SYBR Green，GelRed，虽然毒性小，但价格昂贵。TIANGEN 公司的GeneGreen 相比则是性价比高的低毒替代染料，其灵敏度比传统EB 染料高10 倍以上。注意观察凝胶时应根据染料不同使用合适的光源和激发波长，如果激发波长不对，条带则不易观察，出现条带模糊的现象。
步骤如下：
1. 制备1%琼脂糖凝胶(大胶用70ml，小胶用50ml): 称取0.7 g(0.5 g)琼脂
猎人笔记好句糖置于锥形瓶中，加入70 ml(50ml)1×TAE，瓶口倒扣小烧杯.微波炉加热煮沸 3 次至琼脂糖全部融化，摇匀，即成 1.0%琼脂糖凝胶液.
2. 胶板制备：取电泳槽内的有机玻璃内槽(制胶槽)洗干净，晾干，放入制胶玻璃板. 取透明胶带将玻璃板与内槽两端边缘封好，形成模子.将内槽置于水平位置，并在固定位置放好梳子.将冷却到65℃左右的琼脂糖凝胶液混匀小心地倒入内槽玻璃板上，使胶液缓慢展开，直到整个玻璃板表面形成均匀胶层.室温下静置直至凝胶完全凝固，垂直轻拔梳子，取下胶带，将凝胶及内槽放入电泳槽中.添加1×TAE 电泳缓冲液至没过胶板1-2 ㎜为止.
3. 加样：在点样板或parafilm 上混合DNA 样品和上样缓冲液，上样缓冲液的最终稀释倍数应不小于1X.用10 ul 微量移液器微量移液器分别将样品加入胶板的样品小槽内, 每加完一个样品，应更换一个加样头，以防污染，加样时勿碰坏样品孔周围的凝胶面.(注意：加样前要先记下加样的顺序).
容易脸红
4. 电泳：加样后的凝胶板立即通电进行电泳，电压60-100V，样品由负极(黑色)向正极(红色)方向移动.电压升高，琼脂糖凝胶的有效分离范围降低.当溴酚蓝溴酚蓝移动到溴酚蓝距离胶板下沿约1cm 处时，停止电泳.
儿童游乐项目
5．电泳完毕后，取出凝胶，紫外光下观察。
Agaro Gel Electrophoresis of DNA
Preparing and Running Standard Agaro DNA Gels
The equipment and supplies necessary for conducting agaro gel electrophoresis are relatively simple and include:
发音部位∙An electrophoresis chamber and power supply
∙Gel casting trays, which are available in a variety of sizes and compod of UV-transparent plastic. The open ends of the trays are clod with tape while the gel is being cast, then removed prior to electrophoresis.
∙Sample combs, around which molten agaro is poured to form sample wells in the gel.
∙Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
∙Loading buffer, which contains something den (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.
∙Ethidium bromide, a fluorescent dye ud for staining nucleic acids.
NOTE: Ethidium bromide is a known mutagen and should be handled as
a hazardous chemical - wear gloves while handling.
∙Transilluminator (an ultraviolet lightbox), which is ud to visualize ethidium bromide-stained DNA in gels. NOTE: always wear protective eyewear when obrving DNA on a transilluminator to
prevent damage to the eyes from UV light.
To pour a gel, agaro powder is mixed with electrophoresis buffer to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (final concentration 0.5 ug/ml) at this point to facilitate visualization of DNA after electrophore
sis. After cooling the solution to about 60C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature or, if you are in a big hurry, in a refrigerator.
After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inrted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by obrving bubbles coming off the electrodes. DNA will migrate towards the positive electrode, which is usually colored red.
The distance DNA has migrated in the gel can be judged by visually monitoring migration of the track
ing dyes. Bromophenol blue and xylene cyanol dyes migrate through agaro gels at roughly the same rate as double-stranded DNA fragments of 300 and 4000 bp, respectively.
When adequate migration has occured, DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye intercalates between bas of DNA and RNA. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. Be aware that DNA will diffu within the gel over time, and examination or photography should take place shortly after cessation of electrophoresis.
Migration of DNA Fragments in Agaro
Fragments of linear DNA migrate through agaro gels with a mobility that
of their molecular weight.In other is inverly proportional to the log
10
words, if you plot the distance from the well that DNA fragments have
of either their molecular weights or number of migrated against the log
10
ba pairs, a roughly straight line will appear.
Circular forms of DNA migrate in agaro distinctly differently from linear DNAs of the same mass. Typically, uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two
topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.
Several additional factors have important effects on the mobility of DNA fragments in agaro gels, and can be ud to your advantage in optimizing paration of DNA fragments. Chief among the factors are:好看的发型
Agaro Concentration: By using gels with different concentrations of agaro, one can resolve different sizes of DNA fragments. Higher concentrations of agaro facilite paration of small DNAs, while low agaro concentrations allow resolution of larger DNAs.
>怎么写观察日记
