Sue-Rica

So what you mean is that it doesn't matter what the amplification (RFU) or the Ct value is if there are two curves then it is heterozygous regardless of the intensities. Let say orange represents allele 1 and blue repersents allele 2. On the amplification plot (where you see curves) there are both orange and blue curves but with there two examples: 1: Orange and blue are on top of each other meaning a very clear heterozygote and no difference or space between the two curves, the Ct and RFU's are exactly the same 2: The orange curve is at top and the blue curve just above the threshold, meaning the difference between the two curve are huge Is number two also a heterozygote Example pictures of number 1 and 2 1.bmp 2.bmp

Thank you for replying. My big problem is the Ct values. In theory if you have 2 Ct values one for fam:20.12 and one for vic:20.98 then it is a heterozygote. But where is the cut off Ct value for heterozygotes? We made it a diffirence of +- 1 Ct. If you have an amplification plot with fam having a higher RFU than Vic but the Ct value of vic is higher is it then homozygous for fam or vic?

Hi As I am from South Africa, real time PCR is all new to me. I was very excited to use this new technology on my masters project but with great disapointment. I'm looking at polymorphisms in rare gene so a positive control was not possible at first. Because of this the automatic allelic discrimination software doesn't call work as well. So the big problem is manual genotype calling using the amplification plot. Now this was a big joke. Nowhere can you find info on how to do it. I contacted three reps which included Bio-Rad, Applied Bio and Roche. According to all of them when you get two signals (FAM and VIC) no matter what the amplification of each then you can call it a heterozygote. Is this true. I'm really disappionted with the technology. Even when we tried HRM for diagnotic work on breast cancer the results were inconclusive. Can anyone please help with this. Big question: ARE YOU HAPPY WITH REAL TIME AND IS IT 100% CORRECT?

Hi my I'm new and very excited. I'm from South Africa and need some help with real time PCR. Anyone willing to help?