a****o
发帖数: 2 | 1 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. |
a****o
发帖数: 2 | 2 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. |
l*****2
发帖数: 238 | 3 如果只是最近的问题的话，换酶，换buffer，换PCR machine试试看吧。 |
s*****g
发帖数: 87 | 4 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/ |
