how many cycles needed? - (May/21/2007 )

Hi all

I am new to qPCR and like many others, i have been plagued with problems in my NTC samples. I have read many posts on this issue and i am in the process of trying to determine the cause of this problem, i.e. contamination, primer-dimer etc.. Like many, i have found that i detect SYBR fluroescence in 30+ cycles while my samples are detected from 15 cycles. So my question is... how many cycles do i need?, is it alright to just quantify to 30 cycles?... I have been doing 40 cycles. Just trying to get a general feeling on this.. Thanks for any help

-DanCarter-

Just to be sure - do you find something on the gel in your NTC samples? Generally I would say that everything appearing in real-time RT-PCR above 30cycles is garbage and should not be too much of a worry... if the gel and the melting curves look fine.

-krümelmonster-

Thanks for replying krumel

When i run my reactions on a gel, my NTC has a band at the same size although quite a bit fainter. My melt curve shows a high amplitude peak for the samples at ~85C while my NTC has a low amplitude peak at ~84C. Excuse my inexperience but what does this imply?? Thanks again

-DanCarter-

Hi

Make a master mix with template and all.
Make replicate samoles and run these side by side and remove one set of samples after an additional 5 cycles and run them on a gel.

I did this and saw that I got additional bangs after 33 cycles and heavy problems after 38 and 43 cycles.

However, in fact it doen not matter at all, because you collect your ct values at cycel 15 BEFORE the other bands have been made.

:-)

Peider

-Peider-

QUOTE (DanCarter)

When i run my reactions on a gel, my NTC has a band at the same size although quite a bit fainter.

If you have a band at the expected size in the NTC control, than contamination is an issue. Other possibility - the bands in all wells are primer dimers and not your expected product...

-krümelmonster-