some questions about the component tab of ABI prism 7000 software and the dissoc - (Mar/15/2010 )
hi, everyone. I am running real time PCR with an ABI prism 7000 system. But I have some questions about my result.
The 1st one is about the component tab of the result. I have pasted a pic. I used SYBR Green method with ROX as my reference. But the fluoresce of SYBR Green showed a parabola with the time while ROX remained straight. This is abnormal, right? Can anyone tell me why this happens?
my 2nd question is about the dissociation curve. According to the resulting curve, my PCR product has an Tm about 8 degrees higher than the theoritical value when I designed my primers. And I found my curve has generally a great deviation to the right, compared with those that I have seen done by others. Can anyone help?
fzhang on Mar 15 2010, 09:59 PM said:
The 1st one is about the component tab of the result. I have pasted a pic. I used SYBR Green method with ROX as my reference. But the fluoresce of SYBR Green showed a parabola with the time while ROX remained straight. This is abnormal, right? Can anyone tell me why this happens?
my 2nd question is about the dissociation curve. According to the resulting curve, my PCR product has an Tm about 8 degrees higher than the theoritical value when I designed my primers. And I found my curve has generally a great deviation to the right, compared with those that I have seen done by others. Can anyone help?
Hi!
I think its fine for the ROX signal to be somewhat straight. I would expect the ROX to be stable and being a reference to be somewhat constant and not increase.
And about your second question, if you really think the Tm difference is too high, you could always run a quick gel and make sure that it is the product that you are looking for. Hope this helps.
fzhang on Mar 16 2010, 07:29 AM said:
The 1st one is about the component tab of the result. I have pasted a pic. I used SYBR Green method with ROX as my reference. But the fluoresce of SYBR Green showed a parabola with the time while ROX remained straight. This is abnormal, right? Can anyone tell me why this happens?
my 2nd question is about the dissociation curve. According to the resulting curve, my PCR product has an Tm about 8 degrees higher than the theoritical value when I designed my primers. And I found my curve has generally a great deviation to the right, compared with those that I have seen done by others. Can anyone help?
It is perfectly fine and tat is wat is epected that the ROX is stable and the fluorescence of SYBR green increase!!!!
And if u are talking about the variation withing the theoratical and obtained value, then it can be different as Tm varies with buffer systems too!!
And as kravi rightly suggests u can run a gel or a reference sample (a positive control to ensure that the Tm is fine!!)
Best luck!!