发信人: fingera (dj), 信区: Biology
标 题: Re: PCR primer question
发信站: Unknown Space - 未名空间 (Mon Jul 5 01:26:28 2004), 站内信件

I only use SYBR. What I did is

1. Use Primer Express 1.5 or 2.0 from ABI to design primers. Follow their
guidelines. Or just use Primer3.

2. Use Amply 1.2 (or higher) and Netprimer (http://www.premierbiosoft.com/n
etprimer/) to evaluate candidates.

3. Blast your primers.

4. Pick up 2-3 pairs. Do standard and dissociation curves. Chose the one wor
k best.

Make sure to chose the right normalization primers for your experiment. 18S,
actin, GAPDH, tubulin...


【 在 tootootootoo (tutu) 的大作中提到: 】
: Thank you guys. Big help for me!
: 【 在 shawl (wonderland) 的大作中提到: 】
: : there are 2 way to do QPCR. One is ABI tagman in which the enzyme has a
: : 5'nuclease activity and has a higher specificity. ABI recommend to desigh
: : primer with their software Primer express. Another way is Sybergreen I
: system
: : in which SYBR dye binds minor groove. If you use SYBR, then you have to
: : optimize the annealing temperature for every pair of primer.
: : experience
: are
: fragment
: : design
: : opti-
: PCR.
: my
: : primer
: primer
: : pair
: design
: : 2,


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