qPCR - no signal detected but plenty of product on gel - (Oct/27/2009 )

Hi there,
I did a qPCR reaction using evagreen and i did not get any detection of signal from my transcript of interest. My housekeeping gene controls worked just fine and I got fluorescent signal. I ran my samples on a gel to make sure I didn't leave anything out of my reaction mix. I have bright bands for all of my samples and I can figure out why the signal detection failed. This has happened to me with a different primer pair in the past that was testing another gene transcript all together. I know that I added the evagreen to my mixes, what else could be going on?

Thanks,
Nicole

Nspahich on Oct 28 2009, 12:03 AM said:

weill i will be asuming certain things here... cause evagreen works better than SYBR green acc to many
1) you dint add or prepare different master mixes for your gene of interest and housekeeping genes. If so the dye used for the samples may be corrupt!!!
2) you dint use different dyes for housekeeping and test samples (sorry but dint wanna leave any doubt!!! )
3) worst cum worst check the optical fibres till your detectors for those particular wells.. they might have gone bad leading to no signal.. if the previous case wells match with this one.. do give it a thought!!!
4) Can you attach a melt curve... or you see if tat is fine!!!???

<*>Did you use a positive control? Did that one show up on your machine?
If your + did show up, your primers are probably okay.
If you didn't run a +, you need to.
If your + didn't show up, consider primer issues.

<*>Did your band show up at the right size for your amplicon?
If there is a band, and you detected no product on your pcr machine, the band may be from primers if the size is matched to those.

Nspahich on Oct 27 2009, 07:33 PM said:

did you assign the correct fluoresence markers to you primer pair?
perhaps the computer was looking for fluoresence in a different wavelength for your transcript of interest...