What went wrong in my qPCR? - please look at the dissociation curve (Feb/19/2009 )
The dissociation curve does look weird. Have you run a regular PCR with the primers and your samples? That may give you an idea what is wrong.
Hi all,
Sorry I figured out why it looked very weird.... I did not omit the empty well during analysis... now that I did it, my melting curve still looks weird, but better than before. can you please pour ur suggestions as to why it still looks weird?? please!!!!!!!!! thanks...
A bit overloaded with information, your little picture. Maybe you can toggle of a few samples. Anyway it looks like you have a lot of background fluorescence and primer dimers.
I agree. have you done the titration curves to determine reaction efficiency?
I never trusted plate based real-time from the beginning. I would still go for the rotor based machines like Light Cycler 2 or RotorGene 6000
I've had quite a bit of luck with it in the past, but getting your conditions squared away in the beginning can be a real nightmare.
Curtis on Feb 26 2009, 09:40 AM said:
I know, light cyler or rotorgene is really good and effective.. but our very poor labs have just this plate based systems....!!
![:D](""../../forums/public/style_emoticons/default/sad.gif)
aimikins on Feb 25 2009, 10:21 AM said:
titration curves?? can u please elaborate on how to do that or give me a link which will help me understand the procedure better.... thanks....
Thanks for the response guys...
For now, I am testing my primers on human genomic DNA, hopefully the bands r on the right spot and please god please, no primer dimers!!!!!!!!!!!!!!
PS: I got my primers from the literature, they used these primers in taqman, but I just got the sequences and am using it for SYBR green, did a BLAST search and it returned with just my gene of interest.. Should this be ok???????