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数峰青1989

铁虫 (小有名气)

[求助] 5'race 没有结果,求高人指点

近一段时间在用takara的试剂盒做5'race,但是第二次PCR之后怎么也得不到一个清晰的条带。。。实验结果如所附图,请高人指点一二

RNA分离结果(该结果说明rna提取很成功,可以做下一步实验).jpg



2次PCR之后产物电泳结果(没有条带,原因是什么,请高人指点).jpg
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数峰青1989

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引用回帖:
7楼: Originally posted by platanus at 2013-03-29 22:13:07
反转录之后要用看家基因检测一下反转录的质量的,因为也有可能问题出现在反转录过程的!...

我检测了一下反转录产物的质量,没有任何条带,只有在100bp至300bp左右有弥散的一块。。。这样说一定就是反转录出问题了。。。我再做一下试试
9楼2013-04-01 10:38:05
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数峰青1989

铁虫 (小有名气)

难道没人知道是什么情况么
2楼2013-03-26 21:35:02
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robustli

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【答案】应助回帖

★ ★
小丹木木: 金币+2, 鼓励应助 2013-03-29 16:03:31
gyesang: 回帖置顶 2013-05-31 16:15:43
there are so many reasons such as the quality of the cDNA library, annealing temperature, primer specificity,elongation time. I guess you may need check them one by one. I usually use primer 5 to design my primer, there are other primers design software you can choose. after this try different annealing temperature, sometimes you can add DMSO if the template is GC-rich. At the same time, give it enough extention time. If your target gene is rare, you can also try the nested PCR.
immunologyparasite
3楼2013-03-27 02:02:13
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数峰青1989

铁虫 (小有名气)

引用回帖:
3楼: Originally posted by robustli at 2013-03-27 02:02:13
there are so many reasons such as the quality of the cDNA library, annealing temperature, primer specificity,elongation time. I guess you may need check them one by one. I usually use primer 5 to des ...

谢谢啦,最近的几周里,我做了梯度PCR和巢式PCR,不断的在优化PCR的条件,但是始终得不到结果。于是我就想可能是我自己提取mRNA的过程中出现了问题。我计划再重新提取一遍。不过我有点疑惑:我要提的那个基因的产物是次级代谢产物,我所用的材料是苔藓类植物,我是要选用很嫩的植株,还是选长的时间稍微长一点的植株啊
4楼2013-03-29 15:28:52
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