h*********g
发帖数: 18 | 1 I will design primers for qPCR.
First, I will find the cDNA for the gene from ensemble website. Then put the
cDNA sequence into Primer3 website.
For the parameters, I set as following
Primer size Min 20, Opt 22, Max 27,
Primer Tm, Min 57, Opt 60, Max 63.
GC% Min47, Opt 50, Max 65
It that all I need to consider? Is there anything else I need to pay
attention?
If anyone knows any good websites teaching how to design primers, please let
me know.
Thank you very much. | s******y
发帖数: 28562 | 2 产物不能太长，
应该在50-200bp 之间
最后手工检验一下产物最好不经过一些比较奇怪的地段（比方说超高GC%之类）
the
let
【在 h*********g 的大作中提到】
: I will design primers for qPCR.
: First, I will find the cDNA for the gene from ensemble website. Then put the
: cDNA sequence into Primer3 website.
: For the parameters, I set as following
: Primer size Min 20, Opt 22, Max 27,
: Primer Tm, Min 57, Opt 60, Max 63.
: GC% Min47, Opt 50, Max 65
: It that all I need to consider? Is there anything else I need to pay
: attention?
: If anyone knows any good websites teaching how to design primers, please let
| j****n
发帖数: 3370 | 3 好多网站都有设计软件的吧 比如IDT
the
★ 发自iPhone App: ChineseWeb 8.7
【在 h*********g 的大作中提到】
: I will design primers for qPCR.
: First, I will find the cDNA for the gene from ensemble website. Then put the
: cDNA sequence into Primer3 website.
: For the parameters, I set as following
: Primer size Min 20, Opt 22, Max 27,
: Primer Tm, Min 57, Opt 60, Max 63.
: GC% Min47, Opt 50, Max 65
: It that all I need to consider? Is there anything else I need to pay
: attention?
: If anyone knows any good websites teaching how to design primers, please let
| h*********g
发帖数: 18 | 4 Thanks. What I mean is that are there any other parameters I need to pay
attention to? Such as the product should be around 50 to 200 bp. Are my
parameters correct?
Thanks!
【在 j****n 的大作中提到】
: 好多网站都有设计软件的吧 比如IDT
:
: the
: ★ 发自iPhone App: ChineseWeb 8.7
| A******y
发帖数: 2041 | 5 Just use primerbank, it is really 80-90% to give you a usable RT-primer.
Here we go: http://pga.mgh.harvard.edu/primerbank/ | g*****n
发帖数: 250 | 6 You don't have to worry about the primers given by Primer3 or other online
programs. All the requirements for PCR primers are taken care of. Basically,
more or less 20 nt, Tm 60, GC 50%, no >4 repeats (i.g, AAAAA, GGGG, GCGCGC)
. What you really need to think about is the characteristics of the gene
that you want to test. For example, whether there are alternative spliced
variants and conserved regions with other genes of the same family. | m*********D
发帖数: 1727 | 7 除了上面Artyarty提到的MGH，还有一个成功率很高的site:
http://primerdepot.nci.nih.gov/
IDT网上的那个program似乎不太好，最近按它那个program合成的两个primer sets，
siRNA后居然一个set给出的是2倍的knockdown。换了primer set，同样sample,马上就
是10倍以上的。 |
|
