E*******2
发帖数: 131 | 1 我正在做ChIP-qPCR， anti acetyl Histone H3 and H4, 将来也会考虑做anti
methylation histones，现在遇到的问题
是，找不到合适的control gene primers. Millipore 公司提供的chip kits里用的
GAPDH。 但是我们领域有几篇文章是用
的beta globin和beta tubulin。我的老板说用我们领域已发表的。然后我查了一下他
们用的primer sequences，有意思的
是他们用的是小鼠，但是primer sequences和大鼠的基因是完全匹配的，而和小鼠有1
－2个碱基不匹配。并且beta
globin的primer在大鼠基因中扩增的是hemoglobin epsilone2，而在小鼠中最可能扩增
的是hemoglobin y， beta－like
embryonic chain。 我的问题是，如果我选择beta globin做为我的control gene，我
是否应该重新设计primer，还是说
我可以直接用人家发表的primer sequence？但是他们用的是sybr |
A******y
发帖数: 2041 | 2 If you use taqman, it is unlikely you can use the sybr green primer. There
are RT-PCR primer library these days, just use sybr-green and use the primer
from the library. Also, you need to use multiple house keeping genes these
days. |
E*******2
发帖数: 131 | 3 为什么不行呢？我的之前的目标基因也是用人家用sybr green的primers啊，自己再找
出primer所在的序列设计一个
probe就很好用了啊。
There
primer
these
【在 A******y 的大作中提到】
: If you use taqman, it is unlikely you can use the sybr green primer. There
: are RT-PCR primer library these days, just use sybr-green and use the primer
: from the library. Also, you need to use multiple house keeping genes these
: days.
|
A******y
发帖数: 2041 | 4 Well, I said unlikely, does not mean it is impossible. If you want to take
your time to design the probe, that's fine. As long as you check
amplification linearity with your primers and the probe, you should be okay.
However, you still need multiple housekeeping genes. I have done several
RT-PCR using published primers and they are not linear at all. Do you think
I will really believe their results? |
E*******2
发帖数: 131 | 5 谢谢你，我觉得我把不配对的1，2个碱基更正过来附和小鼠基因顺序应该就可以用了吧
，毕竟两个不同国家的实验室都用
过发表过。另外我学着用相关程序检验了一下，符合我们要求
take
okay.
several
think
【在 A******y 的大作中提到】
: Well, I said unlikely, does not mean it is impossible. If you want to take
: your time to design the probe, that's fine. As long as you check
: amplification linearity with your primers and the probe, you should be okay.
: However, you still need multiple housekeeping genes. I have done several
: RT-PCR using published primers and they are not linear at all. Do you think
: I will really believe their results?
|
m***o
发帖数: 272 | 6 楼主不知道是需要negative control 还是positive control. 最近查到ucdavis的
protocal上有些control primer 的信息。这个protocal感觉还是挺有用。讲的很详细
。希望有用。
Link：http://www.genomecenter.ucdavis.edu/expression_analysis/images/ChIP_Protocol_070702.pdf
Primers Info
+ Control Pol2 F 5’ AGATGAAACCGTTGTCCAAACT 3’
+ Control Pol2 R 5’ AGGTTACGGCAGTTTGTCTCTC 3’
- Control DHFR3’ UTR F 5’ CTGATGTCCAGGAGGAGAAAGG 3’
- - Control DHFR3’ UTR R 5’ AGCCCGACAATGTCAAGGACTG 3’ |
m***o
发帖数: 272 | 7 just found out these sequence is human gene sequence. |
E*******2
发帖数: 131 | 8 谢谢ls的，我positive和negative的都需要。因为所查的文献用的就是这两个基因，所
以怕以后发文章的时候reviewer罗里吧唆，所以决定用这两个的。我后来给公司写信订
primers的时候问清楚了，所以问题应该算解决了。不过你说的这个网站很有用，学习
了。谢谢！ |
