Southern blot实验方法与步骤

?

Southern blot 实验方法与步骤

用于检测重组 DNA, 也可分析 DNA 样品中是否有与探针序列同源的 DNA 片段。 用于基因诊断, 也可验证检测片段的分子量大小。将基因组 DNA 经限制性内切酶酶切,进行琼脂糖电泳,把 分离后定位在凝胶上的不同分子量的 DNA 经碱变性处理, 将凝胶中变性的 DNA 转移至一固相 支持滤膜。利用标记的某一 DNA、RNA 或寡核苷酸与固着于滤膜上的 DNA 发生同源性杂交, 利用放射自显影(化学发光法或显色法)检测。 (一) 器材:台式离心机,恒温水浴锅,电泳仪,水平电泳槽,杂交炉,杂交袋,尼龙膜 或硝酸纤维素膜,转印迹装置,滤纸,吸水纸,紫外交联仪或 80℃烤箱,摇床,X 线胶片。 (二) 试剂:限制性内切酶,DNA 加样缓冲液,DNA Ladder Marker,0.25M HCl,变性液 (0.5M NaOH,1.5M NaCl),中和液(0.5M Tris- HCl PH7.5,3M NaCl), 转印迹液 20?wSSC (3M NaCl,0.3M 柠檬酸钠,PH7.0),标记探针, 2?wSSC,预杂交液和杂交液,2×洗液 (2×SSC,0.1%SDS),0.5×洗液(0.5×SSC,0.1%SDS),1×缓冲洗液(0.1M 马来酸,0.15M NaCl ,PH7.5,0.3%Tween20) ,1×封阻液[1%(W/V)封阻剂溶于 1×马来酸溶液 (0.1M 马来酸, 0.15M NaCl ,PH7.5)],1×检测液(0.1M Tris- HCl,0.1M NaCl,PH7.5),抗-地高辛- 碱性磷酸酶。 注意: 若基因组 DNA 过低, 要以较大体积进行限制酶消化, 消化完毕后, 可以通过乙醇沉淀、 浓缩 DNA 片段,加少量 DNA 加样缓冲液点样。 2、 消化好的样品点样于 0.7%琼脂糖凝胶进行电泳; 注意:为保证 DNA 均匀分散于加样孔,应缓慢将样品加至加样孔。 3、 电泳结束后,将凝胶依次用如下试剂处理进行碱变性,室温下轻轻摇动确保溶液覆盖凝 胶 0.25M HCl 10-15 min 蒸馏水摇洗 5 min 变性液 40 min 蒸馏水摇洗 5 min 中和液 15 min,2 次; 4、 在凝胶碱变性的同时,制备转印迹装置。Southern blot 转膜技术有三种:1),毛细 管转移法; , 2) 电转移法; , 3) 真空转移法。 这里介绍最经典的毛细管转移法 (向上转移) , 在转印迹槽中,倒入 20?wSSC 溶液,槽中置一固相支持物,在固相支持物上从下向上依次 置入:两张与凝胶等宽的滤纸,将滤纸纵向自固相支持物垂于转印迹槽中(简称“桥”), 底面在上的凝胶,滤膜(与凝胶等大),滤纸(与凝胶等大),吸水纸(略小于滤纸,5- 8cm 高),400-800g 重物。凝胶四周用 Parafilm 膜包围防止短路。滤膜事先用 2?wSSC 浸湿至少 5 min。滤纸事先用 20?wSSC 浸湿。转膜 4-18 小时; 注意:转膜装置中各层滤纸和膜之间要将气泡赶净。一旦建立转膜系统后,要防止滤膜和凝 胶错位。防止吸水纸倒塌和完全湿透,要及时更换吸水纸。 5、 转膜结束后,取出滤膜,边角剪一小角做标记。滤膜于 2?wSSC 摇洗 5min,用滤纸吸 干;6、 用紫外交联照射(正面朝上)或于 80℃烤箱烘烤 0.5-2 小时; 7、 膜可立即进行预杂交和杂交,或保存于 4℃待以后应用; 8、 预杂交和杂交 1) 将杂交膜浸湿于 6?wSSC 2min,同时预热预杂交液和杂交炉至预杂交温度; 2 2) 杂交膜封于杂交袋,按 0.2ml/cm 膜面积加入预杂交液,预杂交至少 1 小时; 3) 用于 southern blot 的探针可分为 DNA 探针、RNA 探针和寡核苷酸探针。对探针的标记 方法又可以分为放射性标记和非放射性标记。 这里主要介绍非放射性地高辛标记探针的杂交

方法。杂交时各种探针的用量:DNA 探针,5-25ng/ml;RNA 探针,100ng/ml;寡核苷酸探 针, 0.1-10pmol/ml。 双链 DNA 探针提前 100℃变性 10min 后迅速冰浴, 单链探针无需变性。 将处理后的探针加入杂交液温浴至杂交温度。杂交温度的选择根据杂交液的不同而不同; 注意:应用寡核苷酸探针时,杂交温度的选择。Tm=4?w(G C) 2?w(A T),杂交温度比 Tm 低约 10℃。 4) 弃去预杂交液,将含探针的杂交液注入杂交袋,至少 3.5ml/10?w1℃m,置入杂交炉滚 动; 9、 杂交后的洗膜处理过程,顺序如下: 2×洗液 2×15min 0.5×洗液 2×15min 1×缓冲洗液 1×3min 1×封阻液 1×60min 抗体液* 1×30min 1×缓冲洗液 2×15min 1×检测液 1×5min * 抗-地高辛-碱性磷酸酶用 1×封阻液稀释 10,000 倍(若用显色法稀释 5000 倍;若用 CDP-Star 检测用 20,000 倍稀释); 10、将膜浸于 CSPD 液(CSPD 用 1×检测液稀释 100 倍,CDP-Star 也用 1× 检测液稀释 100 倍)室温避光静置 5min;回收剩余的 CSPD 液或 CDP-Star 液避光保存于 4℃可反复应用 3 -5 次; 11、 将膜上残液吸净, 用保鲜膜封好于 37℃反应 15min, 然后将膜固定于压片夹, 进行曝光。 注意:若用显色剂检测,新鲜配置显色剂(45μlNBT 和 35μl BCIP 溶于 10ml 1×检测液), 将膜浸于显色剂中避光反应 4-16 小时,反应期间不要移动膜。 1. Run the gel as normal. Often for genomic southerns it is desirable to run long gels (18cm) over 4-6hrs. 2. Photograph the gel with a ruler adjacent to the molecular weight markers as a reference. 3. Alkali transfer buffer = 0.5M NaOH (20g/lt), 1.5M NaCl (87.66g/lt). Prepare 1 litre for one gel and another 750ml for each additional gel. BEWARE This buffer is very dangerous, capable of causing severe eye damage. Use the large volumes involved in this procedure with care and wear protective glasses. 4. Add the gel to 250ml alkali transfer buffer, plus 125ml for each additional gel. 5. Place gel on rocker with buffer solution for 20mins. NOTE low % agarose gels must be agitated slowly to prevent tearing. 6. Keep remaining 500ml for the transfer tank. Wear gloves for the following steps 7. Cut 2 pieces of large 3MM paper (wicks) and 2 pieces about 2mm smaller than the gel on each edge and one piece of nylon (Hybond N , Amersham) the same size as the gel. Large gel (HFI) Medium gel Mini gel 2 (14 x 19cm) 2 (15.2 x 10) 2 (9 x 5.2) 2 (14 x 32cm) 2 (15.2 x 32) 2 (18.5 x 5.2) Cut a stack of paper toweling about 2mm smaller than the gel. The stack needs to be

about 6cm thick. 8. Prewet nylon in DDW, then soak in alkali transfer buffer. 9. Add buffer to transfer tank to the level of the platform. Wet the long wicks in transfer buffer and place in the tank. 10. Place gel on platform and spoon on transfer buffer. Add the Nylon and smooth out any bubbles with the back of your finger. 11. Add the 2 slightly smaller 3MM filters, the first prewetted, the second dry. 12. Add the stack of paper towels. NOTE it is very important that the edges of the towel do not touch the wicks that the gel is sitting on or the transfer will be " shortcircuited". 13. Top the stack with a glass or plastic plate and a weight (a bottle with about 200-300ml H2O is ideal). Too much weight compresses the gel and terminates the transfer early. 14. Allow to transfer o/n and then remove the stack carefully. Mark the position of the wells on the filter with a biro (and note position of well #1) before removing the filter. Soak the filter in 0.5M Tris pH 7.5, 1.5M NaCl for 5min. after removal from the gel. 15. Add filter to 200ml 2 X SSC and allow to soak without agitation for 5'. 16. Remove filter and blot dry and bake at 80? for 2hr or place in autoclave for 10' when not operating but warm. 17. Prehybridize with 10ml Aqua. hyb. (Reagents), 1% SDS and 100ug/ml boiled herring sperm DNA for 20-30 minutes (cloned DNA southern) to several hrs (genomic southern). 18. Boil probe for 3' and add to 3ml of fresh hyb solution/SDS/Herring DNA. Squeeze prehyb from the bag and add probe. Seal, avoiding air bubbles and distribute the probe well. Incubate for 4-6hr (cloned DNA) to 16-20hr (genomic southern). Washes are performed in 2 to 0.2X SSC, 0.1% SDS depending on homology of probe to target


相关文档

Southern blot实验方法和操作步骤
Southern Blot原理及实验方法
southern杂交实验方法和步骤
southern blot 原理及试验方法
Southern Blot 实验流程(包括原理细节)
Southern blot实验报告
Northern blot实验步骤
Western blot实验步骤
Western blot 试剂配制及实验步骤
Western Blot 实验过程
电脑版