I'm doing qPCR reaction using SYBEr green master mix Takara, When I did the reaction for the first time, I've got a good result but when I repeated it I got a strange plot that I can't explain. So it'll be great if you can give me any explanation for this plot.
Hi! I saw your protocol. The thermal cycler is fine but there are some differences at melting temperature analysis step compared to my protocol. In my case, after thermal cycler processing the temperature should be set to be increasing from 60 to 90 celsius and ramping time is 0.1 celsius per second.
Seems like some problem with your qPCR system you are using. This is really unexpected, What about your samples?? Are you using one-step or two-step process?
Sometimes, your RNA goes bad unexpectedly when you use one-step technology so repeat experiments performs badly.
Check out the raw readings (Rn, not Delta-Rn, and manybe also the spectral data from which the Rn values are calculated; you get it by the export-function). It could be a problem with the measurement and/or the data processing.
Furher questions may help to isolate the cause of your problem:
Have you checked the PCR products on a gel?
Does the lot of the mastermix work with other samples/primers?
Do other mixes show the same problem on your instrument?
Thanks very much for your help. The problem was in the software. it was setup as Rox while I didn't use it
- Badges
- Science method