dna电泳条带 如何跑得更开

dna电泳条带 如何跑得更开

我用了天根的胶回收试剂盒,回收的DNA 量非常的少最多的一个才9ng/?l 左右,我加了 50ul 的洗脱液.这个到底是出了什么问题呢?我回收的是下面那 3 条跑开的条带,大小是500Bp 左右,下面小的Marker 都跑没了,不知道是为什么,以前都有的,胶是1.2%的浓度,电压120V,时间是25 分钟.建议用原模板进行PCR,如果担心PCR 量较少,可同时扩增几管（最后回收成1 管）,如果 PCR 产物较纯（无非特异性杂带）,则可以直接采用纯化,相对于胶回收,可以减少损失.如果PCR 产物可能存在突变,混在一起后会比较麻烦,你可以就一管回收下来,先连T 克隆,在进行后续载体的构建.PS:做T 克隆时,回收产物只要有就够了,不需要太多的回收产物,即使你回收后的产物电泳检测都看不到都没关系,T 克隆很容易的.关于胶回收问题,我不知道你的片段大概多少,用的什么公司的回收试剂盒.For SOE-PCR:1,never use the SOE-PCR product as the 2ndary PCR template,since it usually contains several different length products which will cause your final products as a smear.2,use about 1/10 conc.of middle primers to increase the PCR products:for example of your case:you should have 3 pairs of PCR primers to amplify the first group products (those three bands on the right side),let's name those primers as 1 to 6.1 and 2 for the 2nd band 3 and 4 for the 3rd band 5 and 6 for the 4th band in this case,odd number primers are forward primers,and even number primers are reverse primers.And let's say you want to PCR them together as the order of 5'-2nd band-3rd band-4th band-3'.When you perform SOE-PCR,in addition of your regular primer 1 and 6 as the pair,add 1/10 the conc.3 and 4 with it.3,Run 2% gel,which will increase the resolution of DNA band (