各位大侠帮帮忙。 我们小组是做土壤微生物的。最近我们组的qPCR 和PCR 的质量都不理想,两个assays 跑出来的gel都很模糊。Both assays give smeary bands in agarose gel, and it sometimes happens to have double bands for qPCR. This problem used to occurred quite a long time ago. And some of the assays are quite routine in the lab. Our group discuss this problem before, and cannot solve it. Anyone can give recommendations? Many thanks. Very urgent.
忘了补充两点 the PCR products for making qPCR standard also had smeary bands. I had tried my best to minimized it by nested PCR with low concentration of DNA template. But I assume certain unspecific amplifications still remained in the qPCR standard, which could lead to smeary bands for the samples of qPCR standard. It however does not explain the existence of smeary bands for the tested samples. Another thing it the Ct value is smaller than usual.
s*h
7 楼
会问工作经历,都干过什么。我的经验是没有问专业问题。
【在 s*******n 的大作中提到】 : 没人知道吗?只能自己顶了 : 还要谢一次
g*s
8 楼
如果,转family 被封的几率有多大 我来试试吧
【在 q***s 的大作中提到】 : 罪与罚(二)
l*2
9 楼
如果只是最近的问题的话,换酶,换buffer,换PCR machine试试看吧。
s*n
10 楼
谢谢啊,赶紧去准备~
【在 s*******h 的大作中提到】 : 会问工作经历,都干过什么。我的经验是没有问专业问题。
B*e
11 楼
汇报一下战果?
【在 g***s 的大作中提到】 : 如果,转family : 被封的几率有多大 : 我来试试吧
s*g
12 楼
For qPCR, primers need to be specially designed to avoid the formation of primer dimer and smear. you can try the primer designing tool from IDT and redesign your qPCR primers, which are the most important thing for a successful qPCR. http://www.idtdna.com/scitools/Applications/RealTimePCR/