发信人: glider (大苹果), 信区: Biology
标 题: Re: questions about rt-pcr
发信站: Unknown Space - 未名空间 (Tue Mar 30 23:18:23 2004) WWW-POST

I have been using invitrogen 2 step RT kit for a while,
always give me very good result ...

On possible is in your case, your total RNA might
be partially degraded, so that you can only get the
shorter segment, instead of the 1kb product

suggestions:
1) double check your primer design
and PCR condition (TM, Mg conc. pfx? or taq, or pfu?)

2) if everything seems to be correct
be more strict with your RNA extraction
use the ambion RNaseZap to remove environmental factors
(just as much as RNAse free as possible)

3) if still don't work, guess you may want to do a 2 step
RT, if you are lucky, you can even buy some cDNA made from
certain tissue directly to use your primers to the reaction

Good luck!

【 在 biochip (老忘) 的大作中提到: 】
: I've been struggling with rt-pcr for a month but nothing shows up. My
expected
: band is around 1kb. The point is my positive control (genomic DNA) works and
: house keeping gene can be RT and PCR as well(150bp). Even the shorter
: fragment inside is still works(600bp) quite good. I's using the AMV one
step
: kit from promega. the condition is 48� 45min, 95�n, (94�,40sec,
50�,40sec,
: 72�ec) for 30cycles, 72�, 7min for extension. Does anybody have idea about
: that? Thanks a lot for your kindly reply in advance!
:
:


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